Bicyclic modulators of androgen receptor function

ABSTRACT

The invention provides compounds of the formula I  
                 
 
     wherein the substitutents are as described herein.  
     Further provided are methods of using such compounds for the treatment of nuclear hormone receptor-associated conditions, such as age related diseases, for example sarcopenia, and also provided are pharmaceutical compositions containing such compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of application Ser. No.10/209,461, filed Jul. 31, 2002, which claims the benefit of U.S.Provisional Application No. 60/309,059, filed Jul. 31, 2001, which areincorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to bicyclic compounds, methods ofusing such compounds in the treatment of androgen receptor-associatedconditions, such as age related diseases, for example sarcopenia, and topharmaceutical compositions containing such compounds.

BACKGROUND OF THE INVENTION

[0003] Nuclear hormone receptors (NHR's) constitute a large super-familyof structurally-related and sequence-specific gene regulators scientistshave named “ligand dependent transcription factors.” R. M. Evans,Science, 240:889 (1988). The steroid binding NHR's (SB-NHR's) form arecognized subset of the NHR's, including the progesterone receptor(PR), androgen receptor (AR), estrogen receptor (ER), glucocorticoidreceptor (GR) and mineralocorticoid receptor (MR). The conventionalnuclear hormone receptors are generally transactivators in the presenceof ligand, which selectively bind to the NHR in a way that effects genetranscription. In the absence of a corresponding ligand, some of theorphan receptors behave as if they are transcriptionally inert. Others,however, behave as either constitutive activators or repressors. Theseorphan nuclear hormone receptors are either under the control ofubiquitous ligands that have not been identified, or do not need to bindligand to exert these activities.

[0004] The AR is a ligand-activated transcriptional regulatory proteinthat mediates induction of male sexual development and function throughits activity with endogenous androgens. In addition, androgens areassociated with male and female maintenance of muscle mass and strength,bone mass and erythropoiesis. Androgens, such as testosterone, also playan important role in many physiological processes, such asdifferentiation of male internal and external genitalia, development andmaintenance of male secondary sexual characteristics (e.g., thedevelopment of prostate, seminal vesicles, penis, scrotum, skeletalmuscle, redistribution of body fat, stimulation of long bone growth,closure of epiphyses, development of male hair growth pattern andenlargement of larynx), the maintenance of sexual behavior and function(e.g., libido and potency) and spermatogenesis (in man).

[0005] As one ages, the serum androgen concentration in the bodydeclines. The age dependent decline in androgens is associated withchanges in body composition for men and woman, such as lower percentageof muscle mass and an increase in body fat, e.g., sarcopenia. In thisregard, modulation of the AR gene can have an impact on thephysiological effects associated with androgen production. However, theeffectiveness of known modulators of steroid receptors is often temperedby their undesired side-effect profile, particularly during long-termadministration. For example, the administration of synthetic androgenshas been associated with liver damage, prostate cancer, adverse effectson male sexual function and adverse effects associated withcardiovascular and erythropoietic function.

[0006] Numerous synthetically derived steroidal and non-steroidalagonists and antagonists have been described for the members of theSB-NHR family. Many of these agonist and antagonist ligands are usedclinically in man to treat a variety of medical conditions. RU486 is anexample of a synthetic antagonist of the PR, which is utilized as abirth control agent (Vegeto et al., Cell 69: 703-713 (1992)). Flutamideis an example of an antagonist of the AR, which is utilized for thetreatment of prostate cancer (Neri et al, Endo. 91, 427-437 (1972)).Tamoxifen is an example of a tissue specific modulator of the ERfunction, that is used in the treatment of breast cancer (Smigel J.Natl. Cancer Inst. 90, 647-648 (1998)). Tamoxifen can function as anantagonist of the ER in breast tissue while acting as an agonist of theER in bone (Grese et al., Proc. Natl. Acad. Sci. USA 94, 14105-14110(1997)). Because of the tissue selective effects seen for Tamoxifen,this agent, and agents like it, are referred to as tissue selectiveestrogen receptor modulator. In addition to synthetically derivednon-endogenous ligands, non-endogenous ligands for NHR's can be obtainedfrom food sources (Regal et al., Proc. Soc. Exp. Biol. Med. 223, 372-378(2000) and Hempstock et al., J. Med. Food 2, 267-269 (1999)). Theflavanoid phytoestrogens are an example of an unnatural ligand forSB-NHR's that are readily obtained from a food source such as soy(Quella et al., J. Clin. Oncol. 18, 1068-1074 (2000) and Banz et al., J.Med. Food 2, 271-273 (1999)). The ability to modulate thetranscriptional activity of individual NHR by the addition of a smallmolecule ligand, makes them ideal targets for the development ofpharmaceutical agents for a variety of disease states.

[0007] As mentioned above, non-natural ligands can be syntheticallyengineered to serve as modulators of the function of NHR's. In the caseof SB-NHR's, engineering of an unnatural ligand can include theidentification of a core structure which mimics the natural steroid coresystem. This can be achieved by random screening against severalSB-NHR's or through directed approaches using the available crystalstructures of a variety of NHR ligand binding domains (Bourguet et al.,Nature 375, 377-382 (1995), Brzozowski, et al., Nature 389, 753-758(1997), Shiau et al., Cell 95, 927-937 (1998) and Tanenbaum et al.,Proc. Natl. Acad. Sci. USA 95, 5998-6003 (1998)). Differentialsubstitution about such a steroid mimic core can provide agents withselectivity for one receptor versus another. In addition, suchmodifications can be employed to obtain agents with agonist orantagonist activity for a particular SB-NHR. Differential substitutionabout the steroid mimic core can result in the formation of a series ofhigh affinity agonists and antagonists with specificity for, forexample, ER versus PR versus AR versus GR versus MR. Such an approach ofdifferential substitution has been reported, for example, for quinolinebased modulators of steroid NHR in J. Med. Chem., 41, 623 (1999); WO9749709; U.S. Pat. No. 5,696,133; U.S. Pat. No. 5,696,130; U.S. Pat. No.5,696,127; U.S. Pat. No. 5,693,647; U.S. Pat. No. 5,693,646; U.S. Pat.No. 5,688,810; U.S. Pat. No. 5,688,808 and WO 9619458, all incorporatedherein by reference.

[0008] Accordingly, identification of compounds which have goodspecificity for one or more steroid receptors, but which have reduced orno cross-reactivity for other steroid or intracellular receptors, wouldbe of significant value in the treatment of male and female hormoneresponsive diseases. There is, therefore, a need in the art for theidentification of selective modulators of the steroid binding nuclearhormone receptors, particularly non-steroidal, non-toxic tissueselective androgen receptor modulators, which activate the androgenreceptor in skeletal muscle while demonstrating limited or neutraleffect on other androgen responsive (e.g., prostate) tissues.

SUMMARY OF THE INVENTION

[0009] In accordance with illustrative embodiments and demonstratingfeatures of the present invention, compounds are provided which arecapable of modulating the function of a nuclear hormone receptor.Preferably the compounds are selective androgen receptor modulators, andhave the general formula I

[0010] wherein

[0011] R₁ is selected from the group consisting of hydrogen (H), cyano(—CN), nitro (—NO₂), halo, heterocyclo, OR₄, CO₂R₅, CONHR₅, COR₅,S(O)_(m)R₅, SO₂NR₅R₅′, NHCOR₅ and NHSO₂R₅;

[0012] R₂ is selected from the group consisting of hydrogen (H), alkylor substituted alkyl, alkenyl or substituted alkenyl, arylalkyl orsubstituted arylalkyl, CO₂R₅, CONR₅R₅′ and CH₂OR₅;

[0013] R₃ is selected from the group consisting of hydrogen (H), alkylor substituted alkyl, alkenyl or substituted alkenyl, alkynyl orsubstituted alkynyl, cycloalkyl or substituted cycloalkyl, arylalkyl orsubstituted arylalkyl, heterocycloalkyl or substituted heterocycloalkyl,aryl or substituted aryl, heteroaryl or substituted heteroaryl, halo,cyano (—CN), NHCOR₅, NHCO₂R₅, NHCONR₅R₅′, NHSO₂R₅ and OR₄;

[0014] R₄ in each functional group is independently selected from thegroup consisting of hydrogen (H), alkyl or substituted alkyl, CHF₂, CF₃and COR₅;

[0015] R₅ and R₅′ in each functional group are each independentlyselected from the group consisting of hydrogen(H), alkyl or substitutedalkyl, alkenyl or substituted alkenyl, alkynyl or substituted alkynyl,cycloalkyl or substituted cycloalkyl, heterocycloalkyl or substitutedheterocycloalkyl, arylalkyl or substituted arylalkyl, aryl orsubstituted aryl, heteroaryl or substituted heteroaryl and —CN;

[0016] W is selected from the group consisting of (CR₆R₆′)_(m),CHOH(CR₆R₆′)_(m), CO(CR₆R₆′)_(m) and C═NOR₄(CR₆R₆′)_(m);

[0017] R₆ and R₆′ are each independently selected from the groupconsisting of hydrogen (H), alkyl or substituted alkyl, alkenyl orsubstituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl orsubstituted cycloalkyl, arylalkyl or substituted arylalkyl,heterocycloalkyl or substituted heterocycloalkyl, aryl or substitutedaryl, heteroaryl or substituted heteroaryl, halo, cyano (—CN), NHCOR₅,NHCO₂R₅, NHCONR₅R₅′, NHSO₂R₅ and OR₄;

[0018] X is selected from the group consisting of methylene (—CH₂—),oxygen(O), S(O)_(m), NCOR₅, NCO₂R₅, NCONHR₅R₅′ NSO₂NR₅R₅′;

[0019] Y is selected from the group consisting of oxygen(O), sulfur(S)and hydrogen (H₂);

[0020] E is selected from the group consisting of C=Z, CHR₅, SO₂, P(O)R₅and P(O)OR₅;

[0021] Z is selected from the group consisting of oxygen(O), sulfur(S),NH and NR₅;

[0022] A and B are each independently selected from the group consistingof hydrogen (H), halo, cyano (—CN), nitro (—NO₂), alkyl or substitutedalkyl and OR₄;

[0023] m is an integer from 0 to 2; and

[0024] n is an integer of 1 or 2.

[0025] The definition of formula I above is inclusive of all prodrugesters, stereoisomers and pharmaceutically acceptable salts of formulaI.

[0026] Further embodiments of the present invention include compounds ofthe formula Ia

[0027] wherein

[0028] R₁ is selected from the group consisting of hydrogen (H), cyano(—CN), nitro (—NO₂), halo, heterocyclo, OR₄, CO₂R₅, CONHR₅, COR₅,S(O)_(m)R₅, SO₂NR₅R₅′, NHCOR₅ and NHSO₂R₅;

[0029] R₂ is selected from the group consisting of hydrogen (H), alkylor substituted alkyl, alkenyl or substituted alkenyl, arylalkyl orsubstituted arylalkyl, CO₂R₅, CONR₅R₅′ and CH₂OR₅;

[0030] R₃ is selected from the group consisting of hydrogen (H), alkylor substituted alkyl, alkenyl or substituted alkenyl, alkynyl orsubstituted alkynyl, cycloalkyl or substituted cycloalkyl, arylalkyl orsubstituted arylalkyl, heterocycloalkyl or substituted heterocycloalkyl,aryl or substituted aryl, heteroaryl or substituted heteroaryl, halo,cyano (—CN), NHCOR₅, NHCO₂R₅, NHCONR₅R₅ 40 , NHSO₂R₅ and OR₄;

[0031] R₄ in each functional group is independently selected from thegroup consisting of hydrogen (H), alkyl or substituted alkyl, CHF₂, CF₃and COR₅;

[0032] R₅ and R₅′ in each functional group are each independentlyselected from the group consisting of hydrogen(H), alkyl or substitutedalkyl, alkenyl or substituted alkenyl, alkynyl or substituted alkynyl,cycloalkyl or substituted cycloalkyl, heterocycloalkyl or substitutedheterocycloalkyl, arylalkyl or substituted arylalkyl, aryl orsubstituted aryl, heteroaryl or substituted heteroaryl and —CN;

[0033] W is selected from the group consisting of (CR₆R₆′)_(m),CHOH(CR₆R₆′)_(m), CO(CR₆R₆′)_(m) and C═NOR₄(CR₆R₆′)_(m);

[0034] R₆ and R₆′ are each independently selected from the groupconsisting of hydrogen (H), alkyl or substituted alkyl, alkenyl orsubstituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl orsubstituted cycloalkyl, arylalkyl or substituted arylalkyl,heterocycloalkyl or substituted heterocycloalkyl, aryl or substitutedaryl, heteroaryl or substituted heteroaryl, halo, cyano (—CN), NHCOR₅,NHCO₂R₅, NHCONR₅R₅′, NHSO₂R₅ and OR₄;

[0035] X is selected from the group consisting of methylene (—CH₂—),oxygen(O), S(O)_(m), NCOR₅, NCO₂R₅, NCONHR₅R₅′ NSO₂NR₅R₅′;

[0036] Y is selected from the group consisting of oxygen(O), sulfur(S)and hydrogen (H₂);

[0037] Z is selected from the group consisting of oxygen(O), sulfur(S),NH and NR₅;

[0038] A and B are each independently selected from the group consistingof hydrogen (H), halo, cyano(—CN), nitro(—NO₂), alkyl or substitutedalkyl and OR₄;

[0039] m is an integer from 0 to 2; and

[0040] n is an integer of 1 or 2.

[0041] The definition of formula Ia above is inclusive of all prodrugesters, stereoisomers and pharmaceutically acceptable salts of formulaIa.

[0042] The compounds of formula I and formula Ia modulate the functionof nuclear hormone receptors, particularly the androgen receptor, andinclude compounds which are, for example, selective agonists, partialagonists, antagonists or partial antagonists of the androgen receptor.Preferably the compounds of formula I and Ia possess activity asagonists of the androgen receptor and may be used in the treatment ofdiseases or disorders associated with androgen receptor activity, suchas maintenance of muscle strength and function (e.g., in the elderly);reversal or prevention of frailty or age-related functional decline(“ARFD”) in the elderly (e.g., sarcopenia); prevention of catabolic sideeffects of glucocorticoids; prevention and treatment of reduced bonedensity or growth (e.g., osteoporosis and osteopenia); treatment ofchronic fatigue syndrome (CFS); chronic myalgia; treatment of acutefatigue syndrome and muscle loss following elective surgery (e.g.,post-surgical rehabilitation); acceleration of wound healing;accelerating bone fracture repair (such as accelerating the recovery ofhip fracture patients); treatment of wasting secondary to fractures andwasting in connection with chronic obstructive pulmonary disease (COPD),chronic liver disease, AIDS, weightlessness, cancer cachexia, burn andtrauma recovery, chronic catabolic state (e.g., coma), eating disorders(e.g., anorexia) and chemotherapy.

[0043] The present invention provides for compounds of formula I and Ia,pharmaceutical compositions employing such compounds and for methods ofusing such compounds. In particular, the present invention provides apharmaceutical composition comprising a therapeutically effective amountof a compound of formula I, Ia or both, alone or in combination with apharmaceutically acceptable carrier.

[0044] Further, in accordance with the present invention, a method isprovided for preventing, inhibiting or treating the progression or onsetof diseases or disorders associated with nuclear hormone receptors,particularly, the androgen receptor, such as the diseases or disordersdefined above and hereinafter, wherein a therapeutically effectiveamount of a compound of formula I, Ia or both, is administered to amammalian, i.e., human, patient in need of treatment.

[0045] The compounds of the invention can be used alone, in combinationwith other compounds of the present invention, or in combination withone or more other agent(s) active in the therapeutic areas describedherein.

[0046] In addition, a method is provided for preventing, inhibiting ortreating the diseases as defined above and hereinafter, wherein atherapeutically effective amount of a combination of a compound offormula I, Ia or both, and another type of therapeutic agent, isadministered to a human patient in need of treatment.

[0047] Preferred are compounds of formula I having the structure

[0048] Especially preferred are compounds of formula Ib wherein:

[0049] R₁ is —NO₂, —CN or halogen;

[0050] R₂ is hydrogen or C₁-C₃ alkyl;

[0051] R₃ is hydrogen or hydroxyl (—OH); and

[0052] n is an integer of 1 or 2.

DETAILED DESCRIPTION OF THE INVENTION

[0053] The following abbreviations are employed herein:

[0054] Chiralpak®=Trademark of Chiral Technologies, Inc. Eaton, Pa.

[0055] DBU=1,8-diazabicyclo[5.4.0]undec-7-ene

[0056] Diaion® WA21J=polyamine resin from Supelco. Co. Bellefonte, Pa.

[0057] EtOAc=ethyl acetate

[0058] HPLC=high performance liquid chromatography

[0059] MeOH=methanol

[0060] MS or Mass Spec=mass spectrometry

[0061] YMC®=trademark of YMC Co, Ltd., Kyoto, Japan

[0062] AcOH=acetic acid

[0063] Boc=tert-butoxycarbonyl

[0064] Et₃N=triethylamine

[0065] DEAD=diethyl azodicarboxylate

[0066] DMF=N,N-dimethylformamide

[0067] DMPU=1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone

[0068] LDA=lithium diisopropylamide

[0069] KOH=potassium hydroxide

[0070] Pd/C=palladium on activated charcoal

[0071] TFA=trifluoroacetic acid

[0072] THF=tetrahydrofuran

[0073] RT=room temperature

[0074] m.p.=melting point

[0075] min=minute(s)

[0076] h or hr=hour(s)

[0077] L=liter

[0078] mL=milliliter

[0079] μL=microliter

[0080] g=gram(s)

[0081] mg=milligram(s)

[0082] mol=moles

[0083] mmol=millimole(s)

[0084] nM=nanomolar

[0085] The following definitions apply to the terms as used throughoutthis specification, unless otherwise limited in specific instances.

[0086] As used herein, the term “alkyl” denotes branched or unbranchedhydrocarbon chains, preferably having about 1 to about 8 carbons, suchas, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl,tert-butyl, 2-methylpentyl pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl and the like. “Substituted alkyl”includes an alkyl group optionally substituted with one or morefunctional groups which are attached commonly to such chains, such as,hydroxyl, bromo, fluoro, chloro, iodo, mercapto or thio, cyano,alkylthio, heterocyclyl, aryl, heteroaryl, carboxyl, carbalkoyl, alkyl,alkenyl, nitro, amino, alkoxyl, amido, and the like to form alkyl groupssuch as trifluoro methyl, 3-hydroxyhexyl, 2-carboxypropyl,2-fluoroethyl, carboxymethyl, cyanobutyl and the like.

[0087] Unless otherwise indicated, the term “cycloalkyl” as employedherein alone or as part of another group includes saturated or partiallyunsaturated (containing 1 or more double bonds) cyclic hydrocarbongroups containing 1 to 3 rings, including monocyclicalkyl, bicyclicalkyland tricyclicalkyl, containing a total of 3 to 20 carbons forming therings, preferably 3 to 10 carbons, forming the ring and which may befused to 1 or 2 aromatic rings as described for aryl, which includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, cyclodecyl and cyclododecyl, cyclohexenyl,

[0088] “Substituted cycloalkyl” includes a cycloalkyl group optionallysubstituted with 1 or more substituents such as halogen, alkyl, alkoxy,hydroxy, aryl, aryloxy, arylalkyl, cycloalkyl, alkylamido,alkanoylamino, oxo, acyl, arylcarbonylamino, amino, nitro, cyano, thioland/or alkylthio and/or any of the alkyl substituents.

[0089] Unless otherwise indicated, the term “alkenyl” or “lower alkenyl”as used herein by itself or as part of another group refers to straightor branched chain radicals of 2 to 20 carbons, preferably 2 to 12carbons, and more preferably 2 to 8 carbons in the normal chain, whichinclude one or more double bonds in the normal chain, such as vinyl,2-propenyl, 3-butenyl, 2-butenyl, 4-pentenyl, 3-pentenyl, 2-hexenyl,3hexenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 3-octenyl, 3-nonenyl,4-decenyl, 3undecenyl, 4-dodecenyl, 4,8,12-tetradecatrienyl, and thelike. “Substituted alkenyl” includes an alkenyl group optionallysubstituted with 1 or more substituents, namely, halogen, haloalkyl,alkyl, alkoxy, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, amino,hydroxy, heteroaryl, cycloheteroalkyl, alkanoylamino, alkylamido,arylcarbonylamino, nitro, cyano, thiol, alkylthio and/or any of thealkyl subs tituents set out herein.

[0090] Unless otherwise indicated, the term “alkynyl” or “lower alkynyl”as used herein by itself or as part of another group refers to straightor branched chain radicals of 2 to 20 carbons, preferably 2 to 12carbons and more preferably 2 to 8 carbons in the normal chain, whichinclude one or more triple bonds in the normal chain, such as2-propynyl, 3-butynyl, 2-butynyl, 4-pentynyl, 3-pentynyl, 2-hexynyl,3-hexynyl, 2-heptynyl, 3-heptynyl, 4-heptynyl, 3-octynyl, 3-nonynyl,4-decynyl,3-undecynyl, 4-dodecynyl and the like. “Substituted alkynyl”includes an alkynyl group optionally substituted with one or moresubstituents, namely, halogen, haloalkyl, alkyl, alkoxy, alkenyl,alkynyl, aryl, arylalkyl, cycloalkyl, amino, heteroaryl,cycloheteroalkyl, hydroxy, alkanoylamino, alkylamido, arylcarbonylamino,nitro, cyano, thiol, and/or alkylthio, and/or any of the alkylsubstituents set out herein.

[0091] The terms “arylalkyl”, “arylalkenyl” and “arylalkynyl” as usedalone or as part of another group refer to alkyl, alkenyl and alkynylgroups as described above having an aryl substituent. Representativeexamples of arylalkyl include, but are not limited to, benzyl,2-phenylethyl, 3-phenylpropyl, phenethyl, benzhydryl and naphthylmethyland the like.

[0092] Where alkyl groups as defined above have single bonds forattachment to other groups at two different carbon atoms, they aretermed “alkylene” groups and may optionally be substituted as definedabove for “alkyl”.

[0093] The term “halogen” or “halo” as used herein alone or as part ofanother group refers to chlorine, bromine, fluorine, and iodine, withchlorine or fluorine being preferred.

[0094] The term “cyano,” as used herein, refers to a —CN group.

[0095] The term “methylene,” as used herein, refers to a —CH₂— group.

[0096] The term “nitro,” as used herein, refers to a —NO₂ group.

[0097] Unless otherwise indicated, the term “aryl” or “Aryl” as employedherein alone or as part of another group refers to monocyclic andbicyclic aromatic groups containing 6 to 10 carbons in the ring portion(such as phenyl or naphthyl including 1-naphthyl and 2-naphthyl) and mayoptionally include one to three additional rings fused to a carbocyclicring or a heterocyclic ring (such as aryl, cycloalkyl, heteroaryl orcycloheteroalkyl rings

[0098] for example

[0099] “Substituted aryl” includes an aryl group optionally substitutedwith 1 or more functional groups, such as halo, haloalkyl, alkyl,haloalkyl, alkoxy, haloalkoxy, alkenyl, trifluoromethyl,trifluoromethoxy, alkynyl, cycloalkyl-alkyl, cycloheteroalkyl,cycloheteroalkylalkyl, aryl, heteroaryl, arylalkyl, aryloxy,aryloxyalkyl, arylalkoxy, alkoxycarbonyl, arylcarbonyl, arylalkenyl,aminocarbonylaryl, arylthio, arylsulfinyl, arylazo, heteroarylalkyl,heteroarylalkenyl, heteroarylheteroaryl, heteroaryloxy, hydroxy, nitro,cyano, amino, substituted amino wherein the amino includes 1 or 2substituents (which are alkyl, aryl or any of the other aryl compoundsmentioned in the definitions), thiol, alkylthio, arylthio,heteroarylthio, arylthioalkyl, alkoxyarylthio, alkylcarbonyl,arylcarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl,aminocarbonyl, alkylcarbonyloxy; arylcarbonyloxy, alkylcarbonylamino,arylcarbonylamino, arylsulfinyl, arylsulfinylalkyl, arylsulfonylamino orarylsulfonaminocarbonyl and/or any of the alkyl substituents set outherein.

[0100] Unless otherwise indicated, the term “heteroaryl” as used hereinalone or as part of another group refers to a 5- or 6-membered aromaticring which includes 1, 2, 3 or 4 hetero atoms such as nitrogen, oxygenor sulfur and such rings fused to an aryl, cycloalkyl, heteroaryl orheterocycloalkyl ring (e.g. benzothiophenyl, indolyl), and includespossible N-oxides. “Substituted heteroaryl” includes a heteroaryl groupoptionally substituted with 1 to 4 substituents such as any of the alkylsubstituents set out above. Examples of heteroaryl groups include thefollowing:

[0101] and the like.

[0102] The term “heterocyclo”, heterocycle or heterocyclic ring, as usedherein, represents an unsubstituted or substituted stable 5- to7-membered monocyclic ring system which may be saturated or unsaturated,and which consists of carbon atoms and from one to four heteroatomsselected from N, O or S, and wherein the nitrogen and sulfur heteroatomsmay optionally be oxidized, and the nitrogen heteroatom may optionallybe quaternized. The heterocyclic ring may be attached at any heteroatomor carbon atom which results in the creation of a stable structure.Examples of such heterocyclic groups include, but is not limited to,piperidinyl, piperazinyl, oxopiperazinyl, oxopiperidinyl,oxopyrrolidinyl, oxoazepinyl, azepinyl, pyrrolyl, pyrrolidinyl, furanyl,thienyl, pyrazolyl, pyrazolidinyl, imidazolyl, imidazolinyl,imidazolidinyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl,oxazolidinyl, isooxazolyl, isoxazolidinyl, morpholinyl, thiazolyl,thiazolidinyl, isothiazolyl, thiadiazolyl, tetrahydropyranyl,thiamorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone, andoxadiazolyl.

[0103] The term “heterocycloalkyl” as used herein, represents acycloalkyl group (nonaromatic) in which one or more of the carbon atomsin the ring is replaced by a heteroatom selected from O, S or N.“Substituted heterocycloalkyl” includes a heterocycloalkyl groupoptionally substituted with one or more substituents, such as any of thealkyl substituents set out above. Suitable heterocycloalkyls include,for example, piperazinyl, morpholinyl, tetrahydropyranyl,tetrahydrofuranyl, piperidinyl, pyrrolidinyl, oxazolinyl, and the like.

[0104] The compounds of formula I can be present as salts, which arealso within the scope of this invention. Pharmaceutically acceptable(i.e., non-toxic, physiologically acceptable) salts are preferred. Ifthe compounds of formula I have, for example, at least one basic center,they can form acid addition salts. These are formed, for example, withstrong inorganic acids, such as mineral acids, for example sulfuricacid, phosphoric acid or a hydrohalic acid, with strong organiccarboxylic acids, such as alkanecarboxylic acids of 1 to 4 carbon atomswhich are unsubstituted or substituted, for example, by halogen, forexample acetic acid, such as saturated or unsaturated dicarboxylicacids, for example oxalic, malonic, succinic, maleic, fumaric, phthalicor terephthalic acid, such as hydroxycarboxylic acids, for exampleascorbic, glycolic, lactic, malic, tartaric or citric acid, such asamino acids, (for example aspartic or glutamic acid or lysine orarginine), or benzoic acid, or with organic sulfonic acids, such as(C₁-C₄) alkyl or arylsulfonic acids which are unsubstituted orsubstituted, for example by halogen, for example methyl- orp-toluene-sulfonic acid. Corresponding acid addition salts can also beformed having, if desired, an additionally present basic center. Thecompounds of formula I having at least one acid group (for example COOH)can also form salts with bases. Suitable salts with bases are, forexample, metal salts, such as alkali metal or alkaline earth metalsalts, for example sodium, potassium or magnesium salts, or salts withammonia or an organic amine, such as morpholine, thiomorpholine,piperidine, pyrrolidine, a mono, di or tri-lower alkylamine, for exampleethyl, tertbutyl, diethyl, diisopropyl, triethyl, tributyl ordimethyl-propylamine, or a mono, di or trihydroxy lower alkylamine, forexample mono, di or triethanolamine. Corresponding internal salts mayfurthermore be formed. Salts which are unsuitable for pharmaceuticaluses but which can be employed, for example, for the isolation orpurification of free compounds of formula I or their pharmaceuticallyacceptable salts, are also included.

[0105] Preferred salts of the compounds of formula I which contain abasic group include monohydrochloride, hydrogensulfate,methanesulfonate, phosphate or nitrate.

[0106] Preferred salts of the compounds of formula I which contain anacid group include sodium, potassium and magnesium salts andpharmaceutically acceptable organic amines.

[0107] The term “modulator” refers to a chemical compound with capacityto either enhance (e.g., “agonist” activity) or inhibit (e.g.,“antagonist” activity) a functional property of biological activity orprocess (e.g., enzyme activity or receptor binding); such enhancement orinhibition may be contingent on the occurrence of a specific event, suchas activation of a signal transduction pathway, and/or may be manifestonly in particular cell types.

[0108] The term “prodrug esters” as employed herein includes esters andcarbonates formed by reacting one or more hydroxyls of compounds offormula I with alkyl, alkoxy, or aryl substituted acylating agentsemploying procedures known to those skilled in the art to generateacetates, pivalates, methylcarbonates, benzoates and the like.

[0109] Any compound that can be converted in vivo to provide thebioactive agent (i.e., the compound of formula I) is a prodrug withinthe scope and spirit of the invention.

[0110] Various forms of prodrugs are well known in the art. Acomprehensive description of prodrugs and prodrug derivatives aredescribed in:

[0111] a.) The Practice of Medicinal Chemistry, Camille G. Wermuth etal., Ch 31, (Academic Press, 1996);

[0112] b.) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985);

[0113] c.) A Textbook of Drug Design and Development, P.Krogsgaard-Larson and H. Bundgaard, eds. Ch 5, pgs 113-191 (HarwoodAcademic Publishers, 199 1).

[0114] Said references are incorporated herein by reference.

[0115] An administration of a therapeutic agent of the inventionincludes administration of a therapeutically effective amount of theagent of the invention. The term “therapeutically effective amount” asused herein refers to an amount of a therapeutic agent to treat orprevent a condition treatable by administration of a composition of theinvention. That amount is the amount sufficient to exhibit a detectabletherapeutic or preventative or ameliorative effect. The effect mayinclude, for example, treatment or prevention of the conditions listedherein. The precise effective amount for a subject will depend upon thesubject's size and health, the nature and extent of the condition beingtreated, recommendations of the treating physician, and the therapeuticsor combination of therapeutics selected for administration. Thus, it isnot useful to specify an exact effective amount in advance.

[0116] All stereoisomers of the compounds of the instant invention arecontemplated, either in admixture or in pure or substantially pure form.The compounds of the present invention can have asymmetric centers atany of the carbon atoms including any one of the R substituents.Consequently, compounds of formula I can exist in enantiomeric ordiastereomeric forms or in mixtures thereof. The processes forpreparation can utilize racemates, enantiomers or diastereomers asstarting materials. When diastereomeric or enantiomeric products areprepared, they can be separated by conventional methods for example,chromatographic, chiral HPLC or fractional crystallization.

[0117] The compounds of formula I of the invention can be prepared asshown in the following reaction schemes and description thereof, as wellas relevant published literature procedures that may be used by oneskilled in the art. Exemplary reagents and procedures for thesereactions appear hereinafter and in the working Examples.

[0118] As illustrated in Scheme I, compounds of formula I can beprepared from suitably protected intermediates of formula II.Intermediates of formula II can be obtained commercially, can beprepared by methods known in the literature or can be readily preparedby one skilled in the art. Treatment of II with an intermediate offormula III yields an intermediate of formula of IV. The intermediatesof formula III can be obtained, for example, from commercially availableisocyanates, thioisocyanates and carbodiimides or can be readilyprepared by one skilled in the art. The intermediate of formula IV canbe heated with or without the presence of a base, such as DBU ortriethylamine, to yield a compound of formula V. Compounds of formula Vrepresent compounds of formula I wherein Y is O or S and E is C=Z.Compounds of formula V where X═S, can be converted to compounds whereX═S(O)_(m) with an oxidizing agent, such as 3-chloroperoxybenzoic acid.Compounds of formula V where X═NH, can be converted to compounds whereX═NCOR₅, NCO₂R₅, NCONHR₅R₅′ NSO₂NR₅R₅′ by methods known in theliterature or can readily prepared by one skilled in the art.

[0119] Scheme II describes a method for preparing compounds of formula Iwherein a suitably protected intermediate of formula II is treated witha phosgene like reagent of formula Cl-E-Cl in the presence of a base,such as NaHCO₃ or triethylamine, to yield an intermediate of formula VI.The phosgene like intermediates of formula Cl-E-Cl can be obtained fromcommercially available sources, can be prepared by methods known in theliterature or can be readily be prepared by one skilled in the art.Phosgene equivalents such as carbonyldiimidazoles, may alternatively besubstituted for Cl-E-Cl. The intermediate of formula VI can be reactedwith an amine of formula VIL in the presence of a base, such astriethylamine, to give an intermediate of formula VIII. The amineintermediates VII can be obtained from commercially available sources,can be prepared by methods known in the literature or can be readily beprepared by one skilled in the art. Subsequently, the intermediate VIIIcan be converted to a compound of formula IX by heating with or withoutthe presence of a base, such as DBU or triethylamine. The compound offormula IX represents compounds of formula I wherein Y is O or S and Eis C=Z, SO₂, P(O)R₅, or P(O)OR₅.

[0120] Scheme III describes a method for preparing compounds of formulaI wherein an intermediate of formula IIa is protected with anN-protecting group (G), such as Boc, to yield an N-protectedintermediate of formula X. The intermediate of formula X can be treatedwith a reducing agent, such as lithium aluminum hydride, to form analcohol intermediate XI, which can be oxidized to an aldehydeintermediate XII. The aldehyde XII can be reacted with an amine offormula VII (as described in Scheme II) in the presence of a reducingagent, such as sodium triacetoxyborohydride to give an intermediate offormula XIII. Removal of the N-protecting group (such as Boc) can beachieved by treatment of the intermediate of formula XIII with acid,such as hydrochloric acid in dioxane, or trifluoroacetic acid inmethylene chloride, to provide an intermediate of formula XIV. Theintermediate of formula XIV can be treated with reagents Cl-E-Cl (asdescribed in Scheme II) in the presence of a base, such astriethylamine, to provide a compound of formula XV wherein E is C=Z,SO₂, P(O)R₅, P(O)OR₅. Alternatively, intermediate XIV can be treatedwith an aldehyde of formula R₅CHO, to provide a compound of formula XVwherein E is CHR₅. The compound of formula XV represents compounds offormula I wherein Y is H₂ and E is C=Z, SO₂, P(O)R₅, P(O)OR₅ or CHR₅.

[0121] Alternatively, a compound of formula V (as described in Scheme I)wherein Y and Z are oxygen (O) can be converted to an intermediate Va bya reducing agent, such as LiEt₃BH, preferably in a solvent such as THFat low temperature (<−40° C.). The intermediate Va can be subsequentlytreated with Et₃SiH in the presence of boron trifluoride diethyletherate in a solvent such as dichloromethane at low temperature (<0°C.) to furnish a compound of formula XV wherein E is C=Z and Z=O.

[0122] As illustrated in Scheme IV, an intermediate of formula X issaponified to an acid XVI by treatment with a base, such as lithiumhydroxide. The acid XVI can be coupled to an amine VII via a variety ofcoupling reagents, for example, as described in “The practice of PeptideSynthesis” (Spring-Verlag, 2^(nd) Ed., Bodanszky, M. and Bodanszky, A.;1993) to yield an amide intermediate of formula XVII. The intermediateof formula XVII is treated with an aldehyde (R₅CHO) in a suitablesolvent (such as toluene), with or without the presence of a base, suchas K₂CO₃, NaOH or DBU, or a weak acid, such as HOAc orp-toluenesulfulfonis acid, to give a compound of formula XIX. Aldehydesof formula R₅CHO can be obtained from commercially available sources,can be prepared by methods known in the literature or readily preparedby one skilled in the art. The compound of formula XIX representscompounds of formula I wherein Y is O and E is CHR₅. Alternatively, thecompound of formula XIX can be treated with Lawesson reagent or P₂O₅ togive a compound of formula XX, which represents compounds of formula Iwhere Y is S and E is CHR₅.

[0123] As illustrated in Scheme V, a suitably protected compound offormula XXI, which is a compound of formula I wherein R₂ is H, can beconverted to a compound of formula I wherein R₂ a functional group otherthan H, as defined herein, by treatment with a base such as LDA and analkyl halide such as iodomethane, preferably in a solvent such as THF atlow temperatures (e.g., −78° C.) to yield a compound of formula I.

[0124] Scheme VI illustrates a means to produce the compounds of formulaI wherein E is C=Z and Z=N—CN. An intermediate of formula II is treatedwith a substituted cyano-thiourea of formula XXII, in the presence of acoupling agent, for example, as described in “The Practice of PeptideSynthesis” (Spring-Verlag, 2^(nd) Ed., Bodanszky, M. and Bodanszky, A.;1993) to yield an intermediate of formula XXIII. The substitutedcyano-thioureas of formula XXII can be obtained from commerciallyavailable sources, methods known in the literature or can readily beprepared by one skilled in the art. The intermediate of formula XXIIIcan be heated with or without the presence of a base, such as DBU, toyield a compound of formula XXIV, which represents a compound of formulaI wherein E is C═N—CN.

[0125] As illustrated in Scheme VII, proline derivatives of formula XXVIwhere R₃ is H or alkyl can be prepared by treatment of compounds offormula XXV with a base, such as potassium tert-butoxide in an aproticsolvent, such as toluene. Removal of the N-protection group (Cbz or Boc)can be achieved by methods known in the literature (“The Practice ofPeptide Synthesis” (Spring-Verlag. 2^(nd) Ed., Bodanszky, M. andBodanszky, A.; 1993). Compounds of formula XXIX can be prepared fromintermediates XXVII following the reactions described in Scheme I.Alternatively, compounds of formula XXIX can also be prepared bytreatment of intermediates XXVIII with a base, such as DBU in a suitablesolvent such as toluene or dichloromethane.

Utility & Combinations A. Utilities

[0126] The compounds of the present invention modulate the function ofthe nuclear hormone receptors, particularly the androgen receptor, andinclude compounds which are, for example, selective agonists, partialagonists, antagonists or partial antagonists of the androgen receptor(AR). Thus, the present compounds are useful in the treatment ofAR-associated conditions. An “AR-associated condition,” as used herein,denotes a condition or disorder which can be treated by modulating thefunction or activity of an AR in a subject, wherein treatment comprisesprevention, partial alleviation or cure of the condition or disorder.Modulation may occur locally, for example, within certain tissues of thesubject, or more extensively throughout a subject being treated for sucha condition or disorder.

[0127] The compounds of the present invention can be administered toanimals, preferably humans, for the treatment of a variety of conditionsand disorders, including, but not limited to maintenance of musclestrength and function (e.g., in the elderly); reversal or prevention offrailty or age-related functional decline (“ARFD”) in the elderly (e.g.,sarcopenia); treatment of catabolic side effects of glucocorticoids;prevention and/or treatment of reduced bone mass, density or growth(e.g., osteoporosis and osteopenia); treatment of chronic fatiguesyndrome (CFS); chronic myalgia; treatment of acute fatigue syndrome andmuscle loss following elective surgery (e.g., post-surgicalrehabilitation); accelerating of wound healing; accelerating bonefracture repair (such as accelerating the recovery of hip fracturepatients); accelerating healing of complicated fractures, e.g.distraction osteogenesis; in joint replacement; prevention ofpost-surgical adhesion formation; acceleration of tooth repair orgrowth; maintenance of sensory function (e.g., hearing, sight,olefaction and taste); treatment of periodontal disease; treatment ofwasting secondary to fractures and wasting in connection with chronicobstructive pulmonary disease (COPD), chronic liver disease, AIDS,weightlessness, cancer cachexia, burn and trauma recovery, chroniccatabolic state (e.g., coma), eating disorders (e.g., anorexia) andchemotherapy; treatment of cardiomyopathy; treatment ofthrombocytopenia; treatment of growth retardation in connection withCrohn's disease; treatment of short bowel syndrome; treatment ofirritable bowel syndrome; treatment of inflammatory bowel disease;treatment of Crohn's disease and ulcerative colits; treatment ofcomplications associated with transplantation; treatment ofphysiological short stature including growth hormone deficient childrenand short stature associated with chronic illness; treatment of obesityand growth retardation associated with obesity; treatment of anorexia(e.g., associated with cachexia or aging); treatment of hypercortisolismand Cushing's syndrome; Paget's disease; treatment of osteoarthritis;induction of pulsatile growth hormone release; treatment ofosteochondrodysplasias; treatment of depression, nervousness,irritability and stress; treatment of reduced mental energy and lowself-esteem (e.g., motivation/assertiveness); improvement of cognitivefunction (e.g., the treatment of dementia, including Alzheimer's diseaseand short term memory loss); treatment of catabolism in connection withpulmonary dysfunction and ventilator dependency; treatment of cardiacdysfunction (e.g., associated with valvular disease, myocardialinfarction, cardiac hypertrophy or congestive heart failure); loweringblood pressure; protection against ventricular dysfunction or preventionof reperfusion events; treatment of adults in chronic dialysis; reversalor slowing of the catabolic state of aging; attenuation or reversal ofprotein catabolic responses following trauma (e.g., reversal of thecatabolic state associated with surgery, congestive heart failure,cardiac myopathy, burns, cancer, COPD etc.); reducing cachexia andprotein loss due to chronic illness such as cancer or AIDS; treatment ofhyperinsulinemia including nesidioblastosis; treatment ofimmunosuppressed patients; treatment of wasting in connection withmultiple sclerosis or other neurodegenerative disorders; promotion ofmyelin repair; maintenance of skin thickness; treatment of metabolichomeostasis and renal homeostasis (e.g., in the frail elderly);stimulation of osteoblasts, bone remodeling and cartilage growth;regulation of food intake; treatment of insulin resistance, includingNIDDM, in mammals (e.g., humans); treatment of insulin resistance in theheart; improvement of sleep quality and correction of the relativehyposomatotropism of senescence due to high increase in REM sleep and adecrease in REM latency; treatment of hypothermia; treatment ofcongestive heart failure; treatment of lipodystrophy (e.g., in patientstaking HIV or AIDS therapies such as protease inhibitors); treatment ofmuscular atrophy (e.g., due to physical inactivity, bed rest or reducedweight-bearing conditions); treatment of musculoskeletal impairment(e.g., in the elderly); improvement of the overall pulmonary function;treatment of sleep disorders; and the treatment of the catabolic stateof prolonged critical illness; treatment of hirsutism, acne, seborrhea,androgenic alopecia, anemia, hyperpilosity, benign prostate hypertrophy,adenomas and neoplasies of the prostate (e.g., advanced metastaticprostate cancer) and malignant tumor cells containing the androgenreceptor, such as is the case for breast, brain, skin, ovarian, bladder,lymphatic, liver and kidney cancers; cancers of the skin, pancreas,endometrium, lung and colon; osteosarcoma; hypercalcemia of malignancy;metastatic bone disease; treatment of spermatogenesis, endometriosis andpolycystic ovary syndrome; conteracting preeclampsia, eclampsia ofpregnancy and preterm labor; treatment of premenstural syndrome;treatment of vaginal dryness; age related decreased testosterone levelsin men, male menopause, hypogonadism, male hormone replacement, male andfemale sexual dysfunction (e.g., erectile dysfunction, decreased sexdrive, sexual well-being, decreased libido), male and femalecontraception, hair loss, Reaven's Syndrome and the enhancement of boneand muscle performance/strength. The term treatment is also intended toinclude prophylactic treatment.

[0128] In addition, the conditions, diseases, and maladies collectivelyreferenced to as “Syndrome X” or Metabolic Syndrome as detailed inJohannsson J. Clin. Endocrinol. Metab., 82, 727-34 (1997), may betreated employing the compounds of the invention.

B. Combinations

[0129] The present invention includes within its scope pharmaceuticalcompositions comprising, as an active ingredient, a therapeuticallyeffective amount of at least one of the compounds of formula I, alone orin combination with a pharmaceutical carrier or diluent. Optionally,compounds of the present invention can be used alone, in combinationwith other compounds of the invention, or in combination with one ormore other therapeutic agent(s), e.g., an antibiotic or otherpharmaceutically active material.

[0130] The compounds of the present invention may be combined withgrowth promoting agents, such as, but not limited to, TRH,diethylstilbesterol, theophylline, enkephalins, E series prostaglandins,compounds disclosed in U.S. Pat. No. 3,239,345, e.g., zeranol, andcompounds disclosed in U.S. Pat. No. 4,036,979, e.g., sulbenox orpeptides disclosed in U.S. Pat. No. 4,411,890.

[0131] The compounds of the invention may also be used in combinationwith growth hormone secretagogues such as GHRP-6, GHRP-1 (as describedin U.S. Pat. No. 4,411,890 and publications WO 89/07110 and WO89/07111), GHRP-2 (as described in WO 93/04081), NN703 (Novo Nordisk),LY444711 (Lilly), MK-677 (Merck), CP424391 (Pfizer) and B-HT920, or withgrowth hormone releasing factor and its analogs or growth hormone andits analogs or somatomedins including IGF-1 and IGF-2, or withalpha-adrenergic agonists, such as clonidine or serotinin 5-HT_(D)agonists, such as sumatriptan, or agents which inhibit somatostatin orits release, such as physostigmine and pyridostigmine. A still furtheruse of the disclosed compounds of the invention is in combination withparathyroid hormone, PTH(1-34) or bisphosphonates, such as MK-217(alendronate).

[0132] A still further use of the compounds of the invention is incombination with estrogen, testosterone, a selective estrogen receptormodulator, such as tamoxifen or raloxifene, or other androgen receptormodulators, such as those disclosed in Edwards, J. P. et al., Bio. Med.Chem. Let., 9, 1003-1008 (1999) and Hamann, L. G. et al., J. Med. Chem.,42, 210-212 (1999).

[0133] A further use of the compounds of this invention is incombination with progesterone receptor agonists (“PRA”), such aslevonorgestrel, medroxyprogesterone acetate (MPA).

[0134] The compounds of the present invention may be employed alone orin combination with each other and/or other modulators of nuclearhormone receptors or other suitable therapeutic agents useful in thetreatment of the aforementioned disorders including: anti-diabeticagents; anti-osteoporosis agents; anti-obesity agents; anti-inflammatoryagents; anti-anxiety agents; anti-depressants; anti-hypertensive agents;anti-platelet agents; anti-thrombotic and thrombolytic agents; cardiacglycosides; cholesterol/lipid lowering agents; mineralocorticoidreceptor antagonists; phospodiesterase inhibitors; protein tyrosinekinase inhibitors; thyroid mimetics (including thyroid receptoragonists); anabolic agents; HIV or AIDS therapies; therapies useful inthe treatment of Alzheimer's disease and other cognitive disorders;therapies useful in the treatment of sleeping disorders;anti-proliferative agents; and anti-tumor agents.

[0135] Examples of suitable anti-diabetic agents for use in combinationwith the compounds of the, present invention include biguanides (e.g.,metformin), glucosidase inhibitors (e.g., acarbose), insulins (includinginsulin secretagogues or insulin sensitizers), meglitinides (e.g.,repaglinide), sulfonylureas (e.g., glimepiride, glyburide andglipizide), biguanide/glyburide combinations (e.g., Glucovance®),thiazolidinediones (e.g., troglitazone, rosiglitazone and pioglitazone),PPAR-alpha agonists, PPAR-gamma agonists, PPAR alpha/gamma dualagonists, SGLT2 inhibitors, glycogen phosphorylase inhibitors,inhibitors of fatty acid binding protein (aP2) such as those disclosedin U.S. Ser. No. 09/519,079 filed Mar. 6, 2000, glucagon-like peptide-1(GLP-1), and dipeptidyl peptidase IV (DP4) inhibitors.

[0136] Examples of suitable anti-osteoporosis agents for use incombination with the compounds of the present invention includealendronate, risedronate, PTH, PTH fragment, raloxifene, calcitonins,steroidal or non-steroidal progesterone receptor agonists, RANK ligandantagonists, calcium sensing receptor antagonists, TRAP inhibitors,selective estrogen receptor modulators (SERM), estrogen and AP-1inhibitors.

[0137] Examples of suitable anti-obesity agents for use in combinationwith the compounds of the present invention include aP2 inhibitors, suchas those disclosed in U.S. Ser. No. 09/519,079 filed Mar. 6, 2000, PPARgamma antagonists, PPAR delta agonists, beta 3 adrenergic agonists, suchas AJ9677 (Takeda/Dainippon), L750355 (Merck), or CP331648 (Pfizer) orother known beta 3 agonists as disclosed in U.S. Pat. Nos. 5,541,204,5,770,615, 5,491,134, 5,776,983 and 5,488,064, a lipase inhibitor, suchas orlistat or ATL-962 (Alizyme), a serotonin (and dopamine) reuptakeinhibitor, such as sibutramine, topiramate (Johnson & Johnson) oraxokine (Regeneron), a thyroid receptor beta drug, such as a thyroidreceptor ligand as disclosed in WO 97/21993 (U. Cal SF), WO 99/00353(KaroBio) and GB98/284425 (KaroBio), and/or an anorectic agent, such asdexamphetamine, phentermine, phenylpropanolamine or mazindol.

[0138] Examples of suitable anti-inflammatory agents for use incombination with the compounds of the present invention includeprednisone, dexamethasone, Enbrel®, cyclooxygenase inhibitors (i.e.,COX-1 and/or COX-2 inhibitors such as NSAIDs, aspirin, indomethacin,ibuprofen, piroxicam, Naproxen®, Celebrex®, Vioxx®), CTLA4-Igagonists/antagonists, CD40 ligand antagonists, IMPDH inhibitors, such asmycophenolate (CellCept®), integrin antagonists, alpha4 beta-7 integrinantagonists, cell adhesion inhibitors, interferon gamma antagonists,ICAM-1, tumor necrosis factor (TNF) antagonists (e.g., infliximab,OR1384), prostaglandin synthesis inhibitors, budesonide, clofazimine,CNI-1493, CD4 antagonists (e.g., priliximab), p38 mitogen-activatedprotein kinase inhibitors, protein tyrosine kinase (PTK) inhibitors, IKKinhibitors, and therapies for the treatment of irritable bowel syndrome(e.g., Zelmac® and Maxi-K® openers such as those disclosed in U.S. Pat.No. 6,184,231 B1).

[0139] Examples of suitable anti-anxiety agents for use in combinationwith the compounds of the present invention include diazepam, lorazepam,buspirone, oxazepam, and hydroxyzine pamoate.

[0140] Examples of suitable anti-depressants for use in combination withthe compounds of the present invention include citalopram, fluoxetine,nefazodone, sertraline, and paroxetine.

[0141] Examples of suitable anti-hypertensive agents for use incombination with the compounds of the present invention include betaadrenergic blockers, calcium channel” blockers (L-type and T-type; e.g.diltiazem, verapamil, nifedipine, amlodipine and mybefradil), diuretics(e.g., chlorothiazide, hydrochlorothiazide, flumethiazide,hydroflumethiazide, bendroflumethiazide, methylchlorothiazide,trichloromethiazide, polythiazide, benzthiazide, ethacrynic acidtricrynafen, chlorthalidone, furosemide, musolimine, bumetanide,triamtrenene, amiloride, spironolactone), renin inhibitors, ACEinhibitors (e.g., captopril, zofenopril, fosinopril, enalapril,ceranopril, cilazopril, delapril, pentopril, quinapril, ramipril,lisinopril), AT-1 receptor antagonists (e.g., losartan, irbesartan,valsartan), ET receptor antagonists (e.g., sitaxsentan, atrsentan andcompounds disclosed in U.S. Pat. Nos. 5,612,359 and 6,043,265), DualET/AII antagonist (e.g., compounds disclosed in WO 00/01389), neutralendopeptidase (NEP) inhibitors, vasopepsidase inhibitors (dual NEP-ACEinhibitors) (e.g., omapatrilat and gemopatrilat), and nitrates.

[0142] Examples of suitable anti-platelet agents for use in combinationwith the compounds of the present invention include GPIIb/IIIa blockers(e.g., abciximab, eptifibatide, tirofiban), P2Y12 antagonists (e.g.,clopidogrel, ticlopidine, CS-747), thromboxane receptor antagonists(e.g., ifetroban), aspirin, and PDE-III inhibitors (e.g., dipyridamole)with or without aspirin.

[0143] Examples of suitable cardiac glycosides for use in combinationwith the compounds of the present invention include digitalis andouabain.

[0144] Examples of suitable cholesterol/lipid lowering agents for use incombination with the compounds of the present invention include HMG-CoAreductase inhibitors (e.g., pravastatin, lovastatin, atorvastatin,simvastatin, NK-104 (a.k.a. itavastatin, or nisvastatin or nisbastatin)and ZD-4522 (a.k.a. rosuvastatin, or atavastatin or visastatin)),squalene synthetase inhibitors, fibrates, bile acid sequestrants, ACATinhibitors, MTP inhibitors, lipooxygenase inhibitors, cholesterolabsorption inhibitors, and cholesterol ester transfer protein inhibitors(e.g., CP-529414).

[0145] Examples of suitable mineralocorticoid receptor antagonists foruse in combination with the compounds of the present invention includespironolactone and eplerinone.

[0146] Examples of suitable phospodiesterase inhibitors for use incombination with the compounds of the present invention include PDEIIIinhibitors such as cilostazol, and phosphodiesterase-5 inhibitors (PDE-5inhibitors) such as sildenafil.

[0147] Examples of suitable thyroid mimetics for use in combination withthe compounds of the present invention include thyrotropin, polythyroid,KB-130015, and dronedarone.

[0148] Examples of suitable anabolic agents for use in combination withthe compounds of the present invention include testosterone, TRHdiethylstilbesterol, estrogens, β-agonists, theophylline, anabolicsteroids, dehydroepiandrosterone, enkephalins, E-series prostagladins,retinoic acid and compounds as disclosed in U.S. Pat. No. 3,239,345,e.g., Zeranol®; U.S. Pat. No. 4,036,979, e.g., Sulbenox® or peptides asdisclosed in U.S. Pat. No. 4,411,890.

[0149] Examples of suitable HIV or AIDS therapies for use in combinationwith the compounds of the present invention include indinavir sulfate,saquinavir, saquinavir mesylate, ritonavir, lamivudine, zidovudine,lamivudine/zidovudine combinations, zalcitabine, didanosine, stavudine,and megestrol acetate.

[0150] Examples of suitable therapies for treatment of Alzheimer'sdisease and cognitive disorders for use in combination with thecompounds of the present invention include donepezil, tacrine,revastigmine, 5HT6, gamma secretase inhibitors, beta secretaseinhibitors, SK channel blockers, Maxi-K blockers, and KCNQs blockers.

[0151] Examples of suitable therapies for treatment of sleepingdisorders for use in combination with the compounds of the presentinvention include melatonin analogs, melatonin receptor antagonists,ML1B agonists, and GABA/NMDA receptor antagonists.

[0152] Examples of suitable anti-proliferative agents for use incombination with the compounds of the present invention includecyclosporin A, paclitaxel, FK 506, and adriamycin.

[0153] Examples of suitable anti-tumor agents for use in combinationwith the compounds of the present invention include paclitaxel,adriamycin, epothilones, cisplatin and carboplatin.

[0154] Compounds of the present invention may further be used incombination with nutritional supplements such as those described in U.S.Pat. No. 5,179,080, especially in combination with whey protein orcasin, amino acids (such as leucine, branched amino acids andhydroxymethylbutyrate), triglycerides, vitamins (e.g., A, B6, B12,folate, C, D and E), minerals (e.g., selenium, magnesium, zinc,chromium, calcium and potassium), carnitine, lipoic acid, creatine,B-hyroxy-B-methylbutyriate (Juven) and coenzyme Q-10.

[0155] In addition, compounds of the present invention may be used incombination with therapeutic agents used in the treatment of sexualdysfunction, including but not limited to PDE-5 inhibitors, such assildenafil or IC-351.

[0156] Compounds of the present invention may further be used incombination with antiresorptive agents, hormone replacement therapies,vitamin D analogues, elemental calcium and calcium supplements,cathepsin K inhibitors, MMP inhibitors, vitronectin receptorantagonists, Src SH₂ antagonists, vacular —H⁺-ATPase inhibitors,ipriflavone, fluoride, Tibolone, prostanoids, 17-beta hydroxysteroiddehydrogenase inhibitors and Src kinase inhibitors.

[0157] Compounds of the present invention may be used in combinationwith male contraceptives, such as nonoxynol 9 or therapeutic agents forthe treatment of hair loss, such as minoxidil and finasteride orchemotherapeutic agents, such as with LHRH agonists.

[0158] Further, the compounds of the present invention may be used incombination with anti-cancer and cytotoxic agents, including but notlimited to alkylating agents such as nitrogen mustards, alkylsulfonates, nitrosoureas, ethylenimines, and triazenes; antimetabolitessuch as folate antagonists, purine analogues, and pyrimidine analogues;antibiotics such as anthracyclines, bleomycins, mitomycin, dactinomycin,and plicamycin; enzymes such as L-asparaginase; farnesyl-proteintransferase inhibitors; 5α reductase inhibitors; inhibitors of17β-hydroxy steroid dehydrogenase type 3; hormonal agents such asglucocorticoids, estrogens/antiestrogens, androgens/antiandrogens,progestins, and luteinizing hormone-releasing hormone antagonists,octreotide acetate; microtubule-disruptor agents, such as ecteinascidinsor their analogs and derivatives; microtubule-stabilizing agents such astaxanes, for example, paclitaxel (Taxol®), docetaxel (Taxotere®), andtheir analogs, and epothilones, such as epothilones A-F and theiranalogs; plant-derived products, such as vinca alkaloids,epipodophyllotoxins, taxanes; and topiosomerase inhibitors;prenyl-protein transferase inhibitors; and miscellaneous agents such ashydroxyurea, procarbazine, mitotane, hexamethylmelamine, platinumcoordination complexes such as cisplatin and carboplatin; and otheragents used as anti-cancer and cytotoxic agents such as biologicalresponse modifiers, growth factors; immune modulators and monoclonalantibodies. The compounds of the invention may also be used inconjunction with radiation therapy.

[0159] Representative examples of these classes of anti-cancer andcytotoxic agents include but are not limited to mechlorethaminehydrochloride, cyclophosphamide, chlorambucil, melphalan, ifosfamide,busulfan, carmustin, lomustine, semustine, streptozocin, thiotepa,dacarbazine, methotrexate, thioguanine, mercaptopurine, fludarabine,pentastatin, cladribin, cytarabine, fluorouracil, doxorubicinhydrochloride, daunorubicin, idarubicin, bleomycin sulfate, mitomycin C,actinomycin D, safracins, saframycins, quinocarcins, discodermolides,vincristine, vinblastine, vinorelbine tartrate, etoposide, etoposidephosphate, teniposide, paclitaxel, tamoxifen, estramustine, estramustinephosphate sodium, flutamide, buserelin, leuprolide, pteridines,diyneses, levamisole, aflacon, interferon, interleukins, aldesleukin,filgrastim, sargramostim, rituximab, BCG, tretinoin, irinotecanhydrochloride, betamethosone, gemcitabine hydrochloride, altretamine,and topoteca and any analogs or derivatives thereof.

[0160] Preferred member of these classes include, but are not limitedto, paclitaxel, cisplatin, carboplatin, doxorubicin, carminomycin,daunorubicin, aminopterin, methotrexate, methopterin, mitomycin C,ecteinascidin 743, or porfiromycin, 5fluorouracil, 6-mercaptopurine,gemcitabine, cytosine arabinoside, podophyllotoxin or podophyllotoxinderivatives such as etoposide, etoposide phosphate or teniposide,melphalan, vinblastine, vincristine, leurosidine, vindesine andleurosine.

[0161] Examples of anticancer and other cytotoxic agents include thefollowing: epothilone derivatives as found in German Patent No.4138042.8; WO 97/19086, WO 98/22461, WO 98/25929, WO 98/38192, WO99/01124, WO 99/02224, WO 99/02514, WO 99/03848, WO 99/07692, WO99/27890, WO 99/28324, WO 99/43653, WO 99/54330, WO 99/54318, WO99/54319, WO 99/65913, WO 99/67252, WO 99/67253 and WO 00/00485; cyclindependent kinase inhibitors as found in WO 99/24416 (see also U.S. Pat.No. 6,040,321); and prenyl-protein transferase inhibitors as found in WO97/30992 and WO 98/54966; and agents such as those described genericallyand specifically in U.S. Pat. No. 6,011,029 (the compounds of which U.S.patent can be employed together with any NHR modulators (including, butnot limited to, those of present invention) such as AR modulators, ERmodulators, with LHRH modulators, or with surgical castration,especially in the treatment of cancer).

[0162] The above other therapeutic agents, when employed in combinationwith the compounds of the present invention, may be used, for example,in those amounts indicated in the Physicians' Desk Reference (PDR) or asotherwise determined by one of ordinary skill in the art.

[0163] The compounds of the formula I can be administered for any of theuses described herein by any suitable means, for example, orally, suchas in the form of tablets, capsules, granules or powders; sublingually;bucally; parenterally, such as by subcutaneous, intravenous,intramuscular, or intrasternal injection or infusion techniques (e.g.,as sterile injectable aqueous or non-aqueous solutions or suspensions);nasally, including administration to the nasal membranes, such as byinhalation spray; topically, such as in the form of a cream or ointment;or rectally such as in the form of suppositories; in dosage unitformulations containing non-toxic, pharmaceutically acceptable vehiclesor diluents. The present compounds can, for example, be administered ina form suitable for immediate release or extended release. Immediaterelease or extended release can be achieved by the use of suitablepharmaceutical compositions comprising the present compounds, or,particularly in the case of extended release, by the use of devices suchas subcutaneous implants or osmotic pumps. The present compounds canalso be administered liposomally.

[0164] Exemplary compositions for oral administration includesuspensions which can contain, for example, microcrystalline cellulosefor imparting bulk, alginic acid or sodium alginate as a suspendingagent, methylcellulose as a viscosity enhancer, and sweeteners orflavoring agents such as those known in the art; and immediate releasetablets which can contain, for example, microcrystalline cellulose,dicalcium phosphate, starch, magnesium stearate and/or lactose and/orother excipients, binders, extenders, disintegrants, diluents andlubricants such as those known in the art. The compounds of formula Ican also be delivered through the oral cavity by sublingual and/orbuccal administration. Molded tablets, compressed tablets orfreeze-dried tablets are exemplary forms which may be used. Exemplarycompositions include those formulating the present compound(s) with fastdissolving diluents such as mannitol, lactose, sucrose and/orcyclodextrins. Also included in such formulations may be high molecularweight excipients such as celluloses (avicel) or polyethylene glycols(PEG). Such formulations can also include an excipient to aid mucosaladhesion such as hydroxy propyl cellulose (HPC), hydroxy propyl methylcellulose (HPMC), sodium carboxy methyl cellulose (SCMC), maleicanhydride copolymer (e.g., Gantrez), and agents to control release suchas polyacrylic copolymer (e.g. Carbopol 934). Lubricants, glidants,flavors, coloring agents and stabilizers may also be added for ease offabrication and use.

[0165] Exemplary compositions for nasal aerosol or inhalationadministration include solutions in saline which can contain, forexample, benzyl alcohol or other suitable preservatives, absorptionpromoters to enhance bioavailability, and/or other solubilizing ordispersing agents such as those known in the art.

[0166] Exemplary compositions for parenteral administration includeinjectable solutions or suspensions which can contain, for example,suitable non-toxic, parenterally acceptable diluents or solvents, suchas mannitol, 1,3-butanediol, water, Ringer's solution, an isotonicsodium chloride solution, or other suitable dispersing or wetting andsuspending agents, including synthetic mono- or diglycerides, and fattyacids, including oleic acid, or Cremaphor.

[0167] Exemplary compositions for rectal administration includesuppositories which can contain, for example, a suitable non-irritatingexcipient, such as cocoa butter, synthetic glyceride esters orpolyethylene glycols, which are solid at ordinary temperatures, butliquify and/or dissolve in the rectal cavity to release the drug.

[0168] Exemplary compositions for topical administration include atopical carrier such as Plastibase (mineral oil gelled withpolyethylene).

[0169] The effective amount of a compound of the present invention canbe determined by one of ordinary skill in the art, and includesexemplary dosage amounts for an adult human of from about 0.01 to 2000mg of active compound per day, which can be administered in a singledose or in the form of individual divided doses, such as from 1 to 4times per day. It will be understood that the specific dose level andfrequency of dosage for any particular subject can be varied and willdepend upon a variety of factors including the activity of the specificcompound employed, the metabolic stability and length of action of thatcompound, the species, age, body weight, general health, sex and diet ofthe subject, the mode and time of administration, rate of excretion,drug combination, and severity of the particular condition. Preferredsubjects for treatment include animals, most preferably mammalianspecies such as humans, and domestic animals such as dogs, cats and thelike, subject to NHR-associated conditions.

Transactivation Assays

[0170] AR Specific Assay:

[0171] Compounds of the present invention were tested in transactivationassays of a transfected reporter construct and using the endogenousandrogen receptor of the host cells. The transactivation assay providesa method for identifying functional agonists and partial agonists thatmimic, or antagonists that inhibit, the effect of native hormones, inthis case, dihydrotestosterone (DHT). This assay can be used to predictin vivo activity as there is a good correlation in both series of data.See, e.g. T. Berger et al., J. Steroid Biochem. Molec. Biol. 773 (1992),the disclosure of which is herein incorporated by reference.

[0172] For the transactivation assay a reporter plasmid is introduced bytransfection (a procedure to induce cells to take foreign genes) intothe respective cells. This reporter plasmid, comprising the cDNA for areporter protein, such as secreted alkaline phosphatase (SEAP),controlled by prostate specific antigen (PSA) upstream sequencescontaining androgen response elements (AREs). This reporter plasmidfunctions as a reporter for the transcription-modulating activity of theAR. Thus, the reporter acts as a surrogate for the products (mRNA thenprotein) normally expressed by a gene under control of the AR and itsnative hormone. In order to detect antagonists, the transactivationassay is carried out in the presence of constant concentration of thenatural AR hormone (DHT) known to induce a defined reporter signal.Increasing concentrations of a suspected antagonist will decrease thereporter signal (e.g., SEAP production). On the other hand, exposing thetransfected cells to increasing concentrations of a suspected agonistwill increase the production of the reporter signal.

[0173] For this assay, LNCaP and MDA 453 cells were obtained from theAmerican Type Culture Collection (Rockville, Md.), and maintained inRPMI 1640 or DMEM medium supplemented with 10% fetal bovine serum (FBS;Gibco) respectively. The respective cells were transiently transfectedby electroporation according to the optimized procedure described byHeiser, 130 Methods Mol. Biol., 117 (2000), with thepSEAP2/PSA540/Enhancer reporter plasmid. The reporter plasmid, wasconstructed as follows: commercial human placental genomic DNA was usedto generate by Polymerase Cycle Reaction (PCR) a fragment containing theBglII site (position 5284) and the Hind III site at position 5831 of thehuman prostate specific antigen promoter (Accession # U37672), Schuur,et al., J. Biol. Chem., 271 (12): 7043-51 (1996). This fragment wassubcloned into the pSEAP2/basic (Clontech) previously digested withBglII and HindIII to generate the pSEAP2/PSA540 construct. Then afragment bearing the fragment of human PSA upstream sequence betweenpositions −5322 and −3873 was amplified by PCR from human placentalgenomic DNA. A XhoI and a BglII sites were introduced with the primers.The resulting fragment was subcloned into pSEAP2/PSA540 digested withXhoI and BglII respectively, to generate the pSEAP2/PSA540/Enhancerconstruct. LNCaP and MDA 453 cells were collected in media containing10% charcoal stripped FBS. Each cell suspension was distributed into twoGene Pulser Cuvetts (Bio-Rad) which then received 8 □g of the reporterconstruct, and electoporated using a Bio-Rad Gene Pulser at 210 voltsand 960 □Faraday. Following the transfections the cells were washed andincubated with media containing charcoal stripped fetal bovine serum inthe absence (blank) or presence (control) of 1 nM dihydrotestosterone(DHT; Sigma Chemical) and in the presence or absence of the standardanti-androgen bicalutamide or compounds of the present invention inconcentrations ranging from 10⁻¹⁰ to 10⁻⁵ M (sample). Duplicates wereused for each sample. The compound dilutions were performed on a Biomek2000 laboratory workstation.

[0174] After 48 hours, a fraction of the supernatant was assayed forSEAP activity using the Phospha-Light Chemiluminescent Reporter GeneAssay System (Tropix, Inc). Viability of the remaining cells wasdetermined using the CellTiter 96 Aqueous Non-Radioactive CellProliferation Assay (MTS Assay, Promega). Briefly, a mix of atetrazolium compound(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium,inner salt; MTS) and an electron coupling reagent (phenazinemethosulfate; PMS) are added to the cells. MTS (Owen's reagent) isbioreduced by cells into a formazan that is soluble in tissue culturemedium, and therefore its absorbance at 490 nm can be measured directlyfrom 96 well assay plates without additional processing. The quantity offormazan product as measured by the amount of 490 nm absorbance isdirectly proportional to the number of living cells in culture. For eachreplicate the SEAP reading was normalized by the Abs490 value derivedfrom the MTS assay. For the antagonist mode, the % Inhibition wascalculated as:

% Inhibition=100×(1−[average control−average blank/averagesample−average blank])

[0175] Data was plotted and the concentration of compound that inhibited50% of the normalized SEAP was quantified (IC₅₀).

[0176] For the agonist mode % Control was referred as the effect of thetested compound compared to the maximal effect observed with the naturalhormone, in this case DHT, and was calculated as:

% Control=100×average sample−average blank/average control−average blank

[0177] Data was plotted and the concentration of compound that activatesto levels 50% of the normalized SEAP for the control was quantified(EC₅₀).

[0178] GR Specificity Assay:

[0179] The reporter plasmid utilized was comprised of the cDNA for thereporter SEAP protein, as described for the AR specific transactivationassay. Expression of the reporter SEAP protein was controlled by themouse mammary tumor virus long terminal repeat (MMTV LTR) sequences thatcontains three hormone response elements (HREs) that can be regulated byboth GR and PR see, e.g. G. Chalepakis et al., Cell, 53(3), 371 (1988).This plasmid was transfected into A549 cells, which expresses endogenousGR, to obtain a GR specific transactivation assay. A549 cells wereobtained from the American Type Culture Collection (Rockville, Md.), andmaintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS;Gibco). Determination of the GR specific antagonist activity of thecompounds of the present invention was identical to that described forthe AR specific transactivation assay, except that the DHT was replacedwith 5 nM dexamethasone (Sigma Chemicals), a specific agonist for GR.Determination of the GR specific agonist activity of the compounds ofthe present invention was performed as described for the ARtransactivation assay, wherein one measures the activation of the GRspecific reporter system by the addition of a test compound, in theabsence of a known GR specific agonists ligand.

[0180] PR Specific Assay:

[0181] The reporter plasmid utilized was comprised of the cDNA for thereporter SEAP protein, as described for the AR specific transactivationassay. Expression of the reporter SEAP protein was controlled by themouse mammary tumor virus long terminal repeat (MMTV LTR) sequences thatcontains three hormone response elements (HREs) that can be regulated byboth GR and PR. This plasmid was transfected into T47D, which expressesendogenous PR, to obtain a PR specific transactivation assay. T47D cellswere obtained from the American Type Culture Collection (Rockville,Md.), and maintained in DMEM medium supplemented with 10% fetal bovineserum (FBS; Gibco). Determination of the PR specific antagonist activityof the compounds of the present invention was identical to thatdescribed for the AR specific transactivation assay, except that the DHTwas replaced with 1 nM Promegastone (NEN), a specific agonist for PR.Determination of the PR specific agonist activity of the compounds ofthe present invention was performed as described for the ARtransactivation assay, wherein one measures the activation of the PRspecific reporter system by the addition of a test compound, in theabsence of a known PR specific agonists ligand.

[0182] AR Binding Assay:

[0183] For the whole cell binding assay, human LNCaP cells (T877A mutantAR) or MDA 453 (wild type AR) in 96-well microtiter plates containingRPMI 1640 or DMEM supplemented with 10% charcoal stripped CA-FBS(Cocaleco Biologicals) respectively, were incubated at 37° C. to removeany endogenous ligand that might be complexed with the receptor in thecells. After 48 hours, either a saturation analysis to determine theK_(d) for tritiated dihydrotestosterone, [³H]-DHT, or a competitivebinding assay to evaluate the ability of test compounds to compete with[³H]-DHT were performed. For the saturation analysis, media (RPMI 1640or DMEM -0.2% CA-FBS) containing [³H]-DHT (in concentrations rangingfrom 0.1 nM to 16 nM) in the absence (total binding) or presence(non-specific binding) of a 500-fold molar excess of unlabeled DHT wereadded to the cells. After 4 hours at 37° C., an aliquot of the totalbinding media at each concentration of [³H]-DHT was removed to estimatethe amount of free [³H]-DHT. The remaining media was removed, cells werewashed three times with PBS and harvested onto UniFilter GF/B plates(Packard), Microscint (Packard) was added and plates counted in aTop-Counter (Packard) to evaluate the amount of bound [³H]-DHT.

[0184] For the saturation analysis, the difference between the totalbinding and the non-specific binding, was defined as specific binding.The specific binding was evaluated by Scatchard analysis to determinethe K_(d) for [3 H]-DHT. See e.g. D. Rodbard, Mathematics and statisticsof ligand assays: an illustrated guide: In: J. Langon and J. J. Clapp,eds., Ligand Assay, Masson Publishing U.S.A., Inc., New York, pp. 45-99,(1981), the disclosure of which is herein incorporated by reference.

[0185] For the competition studies, media containing 1 nM [³H]-DHT andcompounds of the invention (“test compounds”) in concentrations rangingfrom 10⁻¹⁰ to 10⁻⁵ M were added to the cells. Two replicates were usedfor each sample. After 4 hours at 37° C., cells were washed, harvestedand counted as described above. The data was plotted as the amount of[³H]-DHT (% of control in the absence of test compound) remaining overthe range of the dose response curve for a given compound. Theconcentration of test compound that inhibited 50% of the amount of[³H]-DHT bound in the absence of competing ligand was quantified (IC₅₀)after log-logit transformation. The K_(I) values were determined byapplication of the Cheng-Prusoff equation to the IC₅₀ values, where:$K_{1} = {\frac{{IC}_{50}}{( {1 + {{( {{\,^{3}H} - {DHT}} )/K_{d}}\quad {for}\quad {\,^{3}H}} - {DHT}} }.}$

[0186] After correcting for non-specific binding, IC₅₀ values weredetermined. The IC₅₀ is defined as the concentration of competing ligandneeded to reduce specific binding by 50%. The K_(d)s for [³H]-DHT forMDA 453 and LNCaP were 0.7 and 0.2 nM respectively.

[0187] Human Prostate Cell Proliferation Assay:

[0188] Compounds of the present invention were tested (“test compounds”)on the proliferation of human prostate cancer cell lines. For that, MDAPCa2b cells, a cell line derived from the metastasis of a patient thatfailed castration, Navone et al., Clin. Cancer Res., 3, 2493-500 (1997),were incubated with or without the test compounds for 72 hours and theamount of [³H]-thymidine incorporated into DNA was quantified as a wayto assess number of cells and therefore proliferation. The MDA PCa2bcell line was maintained in BRFF-HPC1 media (Biological Research Faculty& Facility Inc., MD) supplemented with 10% FBS. For the assay, cellswere plated in Biocoated 96-well microplates and incubated at 37° C. in10% FBS (charcoal-stripped)/BRFF-BMZERO (without androgens). After 24hours, the cells were treated in the absence (blank) or presence of 1 nMDHT (control) or with test compounds (sample) of the present inventionin concentrations ranging from 10⁻¹⁰ to 10⁻⁵ M. Duplicates were used foreach sample. The compound dilutions were performed on a Biomek 2000laboratory work station. Seventy two hours later 0.44 uCi. of[³H]-Thymidine (Amersham) was added per well and incubated for another24 h followed by tripsinization, harvesting of the cells onto GF/Bfilters. Micro-scint PS were added to the filters before counting themon a Beckman TopCount.

[0189] The % Inhibition was calculated as:

%Inhibition=100×(1−[average_(control)−average_(blank)/average_(sample)−average_(blank)])

[0190] Data was plotted and the concentration of compound that inhibited50% of the [³H]Thymidine incorporation was quantified (IC₅₀).

[0191] C2C12 Mouse Myoblast Transactivation Assay:

[0192] Two functional transactivation assays were developed to assessthe efficacy of androgen agonists in a muscle cell background using aluciferase reporter. The first assay (ARTA Stable 1) uses a cell line,Stable 1 (clone #72), which expresses the full length rat androgenreceptor but requires the transient transfection of anenhancer/reporter. This cell line was derived from C2C12 mouse moyoblastcells. The second assay (ARTA Stable 2) uses a cell line, Stable 2(clone #133), derived from Stable 1 which expresses both rAR and theenhancer/luciferase reporter.

[0193] The enhancer/reporter construct used in this system ispGL3/2XDR-1/luciferase. 2XDR-1 was reported to be an AR specificresponse element in CV-1 cells, Brown et. al. The Journal of BiologicalChemistry 272, 8227-8235, (1997). It was developed by random mutagenesisof an AR/GR consensus enhancer sequence.

[0194] ARTA Stable 1:

[0195] 1. Stable 1 cells are plated in 96 well format at 6,000cells/well in high glucose DMEM without phenol red (Gibco BRL, Cat. No.:21063-029) containing 10% charcoal and dextran treated FBS (HyClone Cat.No.: SH30068.02), 50 mM HEPES Buffer (Gibco BRL, Cat. No.: 15630-080),1X MEM Na Pyruvate (Gibco BRL, Cat. No.: 11360-070), 0.5XAntibiotic-Antimycotic, and 800 μg/ml Geneticin (Gibco BRL, Cat. No.:10131-035).

[0196] 2. 48 hours later, cells are transfected withpGL3/2XDR-1/luciferase using LipofectAMINE Plus® Reagent (Gibco BRL,Cat. No.: 10964-013). Specifically, 5 ng/well pGL3/2XDR-1/luciferase DNAand 50 ng/well Salmon Sperm DNA (as carrier) are diluted with 5 □l/wellOpti-MEMem media (Gibco BRL, Cat. No.: 31985-070). To this, 0.5 □l/wellPlus reagent is added. This mixture is incubated for 15 minutes at roomtemperature. In a separate vessel, 0.385 μl/well LipofectAMINE reagentis diluted with 5 □l/well Opti-MEM. The DNA mixture is then combinedwith the LipofectAMINE mixture and incubated for an additional 15minutes at room temperature. During this time, the media from the cellsis removed and replaced with 60 □l/well of Opti-MEM. To this is added 10□l/well of the DNA/LipofectAMINE transfection mixture. The cells areincubated for 4 hours.

[0197] 3. The transfection mixture is removed from the cells andreplaced with 90 μl of media as in #1 above.

[0198] 4. 10 μl/well of appropriate drug dilution is placed in eachwell.

[0199] 5. 24 hours later, the Steady-Glo™ Luciferase Assay System isused to detect activity according to the manufacturer's instructions(Promega, Cat. No.: E2520).

[0200] ARTA Stable 2:

[0201] 1. Stable 2 cells are plated in 96 well format at 6,000cells/well in high glucose DMEM without phenol red (Gibco BRL, Cat. No.:21063-029) containing 10% charcoal and dextran treated FBS (HyClone Cat.No.: SH30068.02), 50 mM HEPES Buffer (Gibco BRL, Cat. No.: 15630-080),1X MEM Na Pyruvate (Gibco BRL, Cat. No.: 11360-070), 0.5XAntibiotic-Antimycotic, 800 μg/ml Geneticin (Gibco BRL, Cat. No.:10131-035) and 800 μg/ml Hygromycin P (Gibco BRL, Cat. No.: 10687-010).

[0202] 2. 48 hours later, the media on the cells is removed and replacedwith 90 μl fresh. 10 μl/well of appropriate drug dilution is placed ineach well.

[0203] 3. 24 hours later, the Steady-Glo™ Luciferase Assay System isused to detect activity according to the manufacturer's instructions(Promega, Cat. No.: E2520).

Proliferation Assays

[0204] Murine Breast Cell Proliferation Assay:

[0205] The ability of compounds of the present invention (“testcompounds”) to modulate the function of the AR was determined by testingsaid compounds in a proliferation assay using the androgen responsivemurine breast cell line derived from the Shionogi tumor, Hiraoka et al.,Cancer Res., 47, 6560-6564 (1987). Stable AR dependent clones of theparental Shionogi line were established by passing tumor fragments underthe general procedures originally described in Tetuo, et. al., CancerResearch 25, 1168-1175 (1965). From the above procedure, one stableline, SC114, was isolated, characterized and utilized for the testing ofexample compounds. SC114 cells were incubated with or without the testcompounds for 72 hours and the amount of [3H]-thymidine incorporatedinto DNA was quantified as a surrogate endpoint to assess the number ofcells and therefore the proliferation rate as described in Suzuki et.al., J. Steroid Biochem. Mol. Biol. 37, 559-567 (1990). The SC114 cellline was maintained in MEM containing 10⁻⁸ M testosterone and 2%DCC-treated FCS. For the assay, cells were plated in 96-well microplatesin the maintenance media and incubated at 37° C. On the following day,the medium was changed to serum free medium [Ham's F-12:MEM (1;1, v/v)containing 0.1% BSA] with (antagonist mode) or without (agonist mode)10⁻⁸ M testosterone and the test compounds of the present invention inconcentrations ranging from 10⁻¹⁰ to 10⁻⁵ M. Duplicates were used foreach sample. The compound dilutions were performed on a Biomek 2000laboratory work station. Seventy two hours later 0.44 uCi of[3H]-Thymidine (Amersham) was added per well and incubated for another 2hr followed by tripsinization, and harvesting of the cells onto GF/Bfilters. Micro-scint PS were added to the filters before counting themon a Beckman TopCount.

[0206] For the antagonist mode, the % Inhibition was calculated as:

%Inhibition=100×(1−[average_(sample)−average_(blank)/average_(control)−average_(blank)])

[0207] Data was plotted and the concentration of compound that inhibited50% of the [³H]Thymidine incorporation was quantified (IC₅₀).

[0208] For the agonist mode % Control was referred as the effect of thetested compound compared to the maximal effect observed with the naturalhormone, in this case DHT, and was calculated as:

%Control=100×(average_(sample)−average_(blank))/(average_(control)−average_(blank))

[0209] Data was plotted and the concentration of compound that inhibited50% of the [³H]-Thymidine incorporation was quantified (EC₅₀).

[0210] In Vitro Assay to Measure GR Induced AP-1 Transrepression:

[0211] The AP-1 assay is a cell based luciferase reporter assay. A549cells, which contain endogenous glucocorticoid receptor, were stablytransfected with an AP-1 DNA binding site attached to the luciferasegene. Cells are then grown in RPMI+10% fetal calf serum(charcoal-treated)+Penicillin/Streptomycin with 0.5mg/ml geneticin.Cells are plated the day before the assay at approximately 40000cells/well. On assay day, the media is removed by aspiration and 20 μlassay buffer (RPMI without phenol red+10% FCS(charcoal-treated)+Pen/Strep) is added to each well. At this pointeither 20 μl assay buffer (control experiments), the compounds of thepresent invention (“test compounds”) (dissolved in DMSO and added atvarying concentrations) or dexamethasome (100 nM in DMSO, positivecontrol) are added to each well. The plates are then pre-incubated for15 minutes at 37° C., followed by stimulation of the cells with 10 ng/mlPMA. The plates are then incubated for 7 hrs at 37° C. after which 40 μlluciferase substrate reagent is added to each well. Activity is measuredby analysis in a luminometer as compared to control experiments treatedwith buffer or dexamethasome. Activity is designated as % inhibition ofthe reporter system as compared to the buffer control with 10 ng/ml PMAalone. The control, dexamethasone, at a concentration of ≦10 □Mtypically suppresses activity by 65%. Test compounds which demonstratean inhibition of PMA induction of 50% or greater at a concentration oftest compound of ≦10 □M are deemed active.

[0212] Levator Ani & Wet Prostate Weight Assay AR Agonist Assay:

[0213] The activity of compounds of the present invention as AR agonistswas investigated in an immature male rat model, a recognized test ofanabolic effects in muscle and sustaining effects in sex organs for agiven compound, as described in L. G. Hershberger et al., Proc. Soc.Expt. Biol. Med., 83, 175 (1953); B. L. Beyler et al, “Methods forevaluating anabolic and catabolic agents in laboratory animals”, J.Amer. Med. Women's Ass., 23, 708 (1968); H. Fukuda et al.,“Investigations of the levator ani muscle as an anabolic steroid assay”,Nago Dai. Yak. Ken. Nem. 14, 84 (1966) the disclosures of which areherein incorporated by reference.

[0214] The basis of this assay lies in the well-defined action ofandrogenic agents on the maintenance and growth of muscle tissues andsexual accessory organs in animals and man. Androgenic steroids, such astestosterone (T), have been well characterized for their ability tomaintain muscle mass. Treatment of animals or humans after castrationswith an exogenous source of T results in a reversal of muscular atrophy.The effects of T on muscular atrophy in the rat levator ani muscle havebeen well characterized. M. Masuoka et al., “Constant cell population innormal, testosterone deprived and testosterone stimulated levator animuscles” Am. J. Anat. 119, 263 (1966); Z. Gori et al., “Testosteronehypertrophy of levator ani muscle of castrated rats. I. Quantitativedata” Boll.—Soc. Ital. Biol. Sper. 42, 1596 (1966); Z. Gori et al.,“Testosterone hypertrophy of levator ani muscle of castrated rats. II.Electron-microscopic observations” Boll.—Soc. Ital. Biol. Sper. 42, 1600(1966); A. Boris et al., Steroids 15, 61 (1970). As described above, theeffects of androgens on maintenance of male sexual accessory organs,such as the prostate and seminal vesicles, is well described. Castrationresults in rapid involution and atrophy of the prostate and seminalvesicles. This effect can be reversed by exogenous addition ofandrogens. Since both the levator ani muscle and the male sex organs arethe tissues most responsive to the effects of androgenic agents, thismodel is used to determine the androgen dependent reversal of atrophy inthe levator ani muscle and the sex accessory organs in immaturecastrated rats. Sexually mature rats (200-250 g, 6-8 weeks-old,Sprague-Dawley, Harlan) were acquired castrated from the vendor(Taconic). The rats were divided into groups and treated daily for 7 to14 days with one of the following:

[0215] 1. Control vehicle

[0216] 2. Testosterone Propionate (TP) (3 mg/rat/day, subcutaneous)

[0217] 3. TP plus Casodex (administered p.o. in PEGTW, QD), a recognizedantiandrogen, as a reference compound.

[0218] 4. To demonstrate antagonist activity, a compound of the presentinvention (“test compound”) was administered (p.o. in PEGTW, QD) with TP(s.c. as administered in group 2) in a range of doses.

[0219] 5. To demonstrate agonist activity a compound of the presentinvention (“test compound”) was administered alone (p.o. in PEGTW, QD)in a range of doses.

[0220] At the end of the 7-14-day treatment, the animals were sacrificedby carbon dioxide, and the levator ani, seminal vesicle and ventralprostate weighed. To compare data from different experiments, thelevator ani muscle and sexual organ weights were first standardized asmg per 100 g of body weight, and the increase in organ weight induced byTP was considered as the maximum increase (100%). Super-anova (onefactor) was used for statistical analysis.

[0221] The gain and loss of sexual organ weight reflect the changes ofthe cell number (DNA content) and cell mass (protein content), dependingupon the serum androgen concentration. See Y. Okuda et al., J. Urol.,145, 188-191 (1991), the disclosure of which is herein incorporated byreference. Therefore, measurement of organ wet weight is sufficient toindicate the bioactivity of androgens and androgen antagonist. Inimmature castrated rats, replacement of exogenous androgens increaseslevator ani, seminal vesicles (SV) and prostate in a dose dependentmanner.

[0222] The maximum increase in organ weight was 4 to 5-fold when dosing3 mg/rat/day of testosterone (T) or 1 mg/rat/day of testosteronepropionate (TP) for 3 days. The EC₅₀ of T and TP were about 1 mg and0.03 mg, respectively. The increase in the weight of the VP and SV alsocorrelated with the increase in the serum T and DHT concentration.Although administration of T showed 5-times higher serum concentrationsof T and DHT at 2 hours after subcutaneous injection than that of TP,thereafter, these high levels declined very rapidly. In contrast, theserum concentrations of T and DHT in TP-treated animals were fairlyconsistent during the 24 hours, and therefore, TP showed about10-30-fold higher potency than free T.

[0223] The following examples serve to better illustrate, but not limit,some of the preferred embodiments of the invention.

EXAMPLE 1Tetrahydro-2-(4-nitro-1-naphthalenyl)imidazo[1,5-α]pyridine-1,3(2H,5H)-dione

[0224]

1A. 4-Nitro-1-naphthaleneisocyanate

[0225]

[0226] To a yellow solution of 1-amino-4-nitronaphthalene (5.17 g, 27.5mmol) in CH₂Cl₂ (80 mL) was added solid NaHCO₃ (23.10 g, 275 mmol). Theresulting suspension was stirred at 0° C. for 15 min, then phosgene(20%) in toluene (110 mmol) was added rapidly to the suspension. Afteraddition, the mixture was stirred at RT for 2 hours, then filtered toremove the solid. The filtrate was concentrated under reduced pressure,the resulting solid residue dried in vacuo for 1 hour to giveapproximately 6.0 g of the title compound as a brown solid.

1B.Tetrahydro-2-(4-nitro-1-naphthalenyl)imidazo[1,5-α]pyridine-1,3(2H,5H)-dione

[0227] To a solution of ethyl pipecolinate (0.234 g, 1.49 mmol) andcompound 1A (0.29 g, 1.355 mmol) in toluene (6 mL) was added 4 Åmolecular sieves (1.0 g). The mixture was stirred at room temperatureovernight, filtered. The filtrate was diluted with EtOAc, washed with 1N HCl, water, brine, dried over MgSO₄, concentrated under reducedpressure to give a brow colored foam, which was chromatographed (silicagel) eluting with CH₂Cl₂, 0.3% MeOH in CH₂Cl₂ to give 110 mg of thetitle compound (racemate) as a yellow solid. mp. 190-192° C.; HPLC: 100%at 2.64 min (retention time) (YMC S5 ODS column 4.6×50mm; 10-90% aqueousmethanol over 4 minutes containing 0.2% phosphoric acid, 4 mL/min,monitoring at 220 nM), MS (ES): m/z 326 [M+1]⁺.

EXAMPLE 2Tetrahydro-2-(4-nitro-1-naphthalenyl)-1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione

[0228]

2A.(2S)-1-[[(4-nitro-1-naphthalenyl)amino]carbonyl]-2-pyrrolidinecarboxylicacid methyl ester

[0229]

[0230] To a solution of L-proline methyl ester hydrochloride (257 mg,1.43 mmol) in MeOH was added triethylamine (0.2 mL, 1.43 mmol). Thesolution was concentrated under reduced pressure to give a white solidresidue, which was triturated with ether (3×). The collected ethersolution was concentrated under reduced pressure to give L-prolinemethyl ester free amine (220 mg) as a colorless oil. To a solution ofabove free amine in toluene (5 mL) was added compound 1A (278 mg, 1.3mmol), followed by 4 Å molecular sieves (1.0 g). The mixture was stirredat room temperature overnight and filtered. The filtrate was dilutedwith EtOAc, washed with 1 N aqueous HCl, water, brine, dried over MgSO₄,concentrated under reduced pressure to give a crude product, which waschromatographed (silica gel) eluting with CH₂Cl₂, 0.5% MeOH in CH₂Cl₂ togive the title compound (410 mg, 92% yield) as a yellow foam.

2B.Tetrahydro-2-(4-nitro-1-naphthalenyl)-1H-pyrrolo[1,2c]imidazole-1,3(2H)-dione

[0231] A suspension of compound 2A (410 mg, 1.29 mmol) and DBU (0.2 mL,1.33 mmol) in toluene (10 mL) was heated at 80° C. for 2 hours. HPLCcheck of the reaction showed little product formed. Additional DBU (0.1mL) was added and the reaction heated at 90° C. overnight. After coolingto RT, the reaction mixture was filtered. The filtrate was diluted withEtOAc, washed with 1 N aqueous HCl, water, brine, dried over MgSO₄ andconcentrated under reduced pressure to give a crude product, which waschromatographed (silica gel) eluting with CH₂Cl₂, 0.2% to 0.4% MeOH inCH₂Cl₂ to give the title compound (racemate) (80 mg) as a brown solid.mp. 215-217° C. HPLC: 97.5% at 2.51 min (retention time) (YMC S5 ODScolumn 4.6×50 mm; 10-90% aqueous methanol over 4 minutes containing 0.2%phosphoric acid, 4 mL/min, monitoring at 220 nM), MS (ES): m/z 312[M+1]⁺.

EXAMPLE 3(6S)-Tetrahydro-6-hydroxy-2-(4-nitro-1-naphthalenyl)-1H-pyrrolo[1,2c]imidazole-1,3(2H)-dione

[0232]

[0233] cis-4-Hydroxy-L-proline methyl ester hydrochloride salt (107 mg,0.59 mmol) in MeOH was treated with triethylamine (82 μL). The solutionwas concentrated under reduced pressure and the resulting residuetriturated with ether (3×50 mL). The combined ether extracts wereconcentrated under reduced pressure to give the amino acid ester as afree amine, which was suspended in toluene (10 mL) and compound 1A (126mg, 0.59 mmol) was added, followed by 4 Å molecular sieves (˜1.0 g). Themixture was stirred at RT until compound 1A was consumed (˜2 hours). DBU(90 μL, 0.59 mmol) was added, the resulting dark-red colored suspensionwas heated at 67° C. for 2 hours. The reaction mixture was cooled to RTand filtered. The filtrate was diluted with EtOAc, washed with saturatedaqueous NH₄Cl, water, brine, dried over MgSO₄ and concentrated underreduced pressure to give a crude product, which was chromatographed(silica gel) eluting with 0.2% MeOH in EtOAc/hexane (7:3) to give thetitle compound (140 mg, 73% yield) as a yellow solid. mp. 249-251° C.HPLC: 99% at 2.13 to 2.28 min (retention time) (YMC S5 ODS column4.6×50mm; 10-90% aqueous methanol over 4 minutes containing 0.2%phosphoric acid, 4 mL/min, monitoring at 220 nM), MS (ES): m/z 328[M+1]⁺.

EXAMPLE 4(6R)-Tetrahydro-6-hydroxy-2-(4-nitro-1-naphthalenyl)-1H-pyrrolo [1,2c]imidazole-1,3(2H)-dione

[0234]

[0235] The title compound was prepared from trans-4-hydroxy-L prolinemethyl ester hydrochloride salt by the procedures analogous to thosedescribed in Example 3. mp. 246-248’ C. HPLC: 99% at 2.16 to 2.31 min(retention time) (YMC S5 ODS column 4.6×50mm; 10-90% aqueous methanolover 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at220 nM), MS (ES): m/z 328 [M+1]⁺.

EXAMPLE 5(6R)-Tetrahydro-2-(4-nitro-1-naphthalenyl)-6-(Phenylmethoxy)-1H-pyrrolo]1,2-c]imidazole-1,3(2H)-dione

[0236]

[0237] The title compound was prepared from trans-4-benzyloxy-L prolinemethyl ester hydrochloride salt by the procedures analogous to thosedescribed in Example 3. mp. 120-122° C. HPLC: 99% at 3.307 min(retention time) (YMC S5 ODS column 4.6×50mm; 10-90% aqueous methanolover 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at220 nM), MS (ES): m/z 418 [M+1]⁺.

EXAMPLE 6trans-4-(Tetrahydro-7-hydroxy-1.3-dioxo-1H-pyrrolo[1,2c]-1imidazol-2(3H)-yl)-1-naphthalenecarbonitrile

[0238]

6A. (2S,3S)-3-Hydroxy-2-pyrrolidinecarboxylic acid methyl ester

[0239]

[0240] Hydrogen chloride gas was bubbled through a suspension oftrans-3-hydroxy-L-proline (8 g, 61 mmol) in MeOH (800 mL) cooled at 0°C. for 10 minutes. The resulting clear solution was stirred at RT for 2hours. The reaction mixture was concentrated under reduced pressure, andobtained residue stripped with toluene, dried in vacuo to yield 12 g ofthe product as a HCl salt. To a solution of above ester HCl salt in MeOHwas added 26 g of Diaion® WA21J resin (pre-washed with MeOH). Theresulting suspension was stirred at RT for 30 minutes, then filtered.The filtrate was concentrated under reduced pressure to give the titlecompound as a colorless oil.

6B. 4-Cyano-1-naphthaleneisocyanate

[0241]

[0242] The title compound was prepared from4-amino-1-naphthalenecarbonitrile in a similar fashion as that describedin Example 1A.

6C.trans-4-(Tetrahydro-7-hydroxy-1,3-dioxo-1H-pyrrolo[1,2c]imidazol-2(3H)-yl)-1-naphthalenecarbonitrile

[0243] To a suspension of 6B (5.35 g, 27.5 mmol) in toluene (500 mL) wasadded compound 6A (4.0 g, 27.5 mmol), followed by 4 Å molecular sieves(˜10 g). The resulting mixture was stirred at RT overnight, then DBU(4.2 mL, 27.5 mmol) was added. The resulting dark-red colored suspensionwas heated at 76° C. for 2 hours, then cooled at RT and filtered. Thefiltrate was diluted with EtOAc, washed with saturated aqueous NH₄Cl,water, brine, dried over MgSO₄, concentrated under reduced pressure togive a crude product, which was chromatographed (silica gel) elutingwith EtOAc/hexane (1:1), 0.2 to 0.5% MeOH in EtOAc/hexane (1:1) to give3.8 g of the title compound (racemate) as a beige colored solid. mp.224-225° C. HPLC: 99% at 2.03 min (retention time) (YMC S5 ODS column4.6×50 mm; 10-90% aqueous methanol over 4 minutes containing 0.2%phosphoric acid, 4 mL/min, monitoring at 220 nM), MS (ES): m/z 308[M+1]⁺

EXAMPLE 7(7R,7aS)-4-(Tetrahydro-7-hydroxy-1,3-dioxo-1H-pyrrolo[1,2c]imidazol-2(3H)-yl)-1-naphthalenecarbonitrile

[0244]

[0245] The title compound (1.77 g) was isolated from 6C (5.8 g) by usingpreparative HPLC with a chiral column (CHIRALPAK® AD, 5×50 cm, 20μ) and25% of isopropanol/hexane as an eluate. mp. 229-230° C.; [α]_(D)^(rt)=−29.76; HPLC: 99% at 12.43 min (retention time) (CHIRALPAK® ODcolumn 4.6×250 mm; 30% isopropanol in hexane over 20 minutes, 1 mL/min,monitoring at 220 nM); MS (ES): m/z 308 [M+1]^(+l .)

[0246] The title compound was also prepared by using a homochiralsynthesis described below.

7A. (2S,3S)-N-tert-Butoxycarbonyl-3-hydroxy-2-pyrrolidinecarboxylic acidmethyl ester

[0247]

[0248] Hydrogen chloride gas was bubbled through a suspension oftrans-3-hydroxy-L-proline (10 g, 76 mmol) in MeOH (300 mL) cooled at 0°C. for 10 minutes. The resulting clear solution was stirred at RT for 2hours. The reaction mixture was concentrated under reduced pressure, theresulting residue stripped with toluene and dried in vacuo to yield 14 gof methyl ester hydrochloride salt as a white solid. To a suspension of11 g of the above ester hydrochloride salt in CH₂Cl₂ (300 mL) at RT wasadded Et₃N (16.8 mL), followed by addition of di-tert-butyl dicarbonate(13.2 g) in portions. The mixture was stirred at RT for 3 hours, thenpartitioned between water and CH₂Cl₂. The organic phase was washed withsaturated aqueous NaHCO₃ and brine, dried over Na₂SO₄ and filtered. Thefiltrate was concentrated under reduced pressure to give a crudeproduct, which was chromatographed (silca gel) eluting with 50%EtOAc/hexane to give 13.6 g of the title compound as a colorless oil.

7B. (2S,3R)-N-tert-Butoxycarbonyl-3-benzoyloxy-2-pyrrolidinecarboxylicacid methyl ester

[0249]

[0250] To a solution of 7A (13.5 g, 55 mmol) in anhydrous THF (500 mL)was added triphenylphosphine (26 g, 99 mmol), followed by benzoic acid(10.1 g, 82.7 mmol) and DEAD (9.5 mL, 60 mmol). The reaction mixture wasstirred at RT for 2 hours, then partitioned between water and CH₂Cl₂.The organic phase was washed with saturated aqueous NaHCO₃ and brine,dried over Na₂SO₄ and filtered. The filtrate was concentrated underreduced pressure to give a crude product, which was chromatographed(silica gel) eluting with 15% EtOAc/hexane to give 18.1 g of the titlecompound as a colorless oil.

7C. (2S,3R)-N-tert-Butoxycarbonyl-3-hydroxy-2-pyrrolidinecarboxylic acidmethyl ester

[0251]

[0252] To a solution of 7B (18 g, 51.5 mmol) in anhydrous MeOH (360 mL)at RT was slowly added a freshly prepared 1N solution of KOH inanhydrous MeOH (77 mL, 77 mmol) in portions. The mixture was stirred atRT until the reaction was completed (about 1 hour). The reaction wascooled at 0° C., then quenched by slow addition of 1N aqueous HCl (77mL). The mixture was concentrated under reduced pressure to remove mostof the MeOH solvent, and the remaining mixture partitioned between waterand EtOAc. The separated EtOAc phase was washed with saturated aqueousNaHCO₃ and brine, dried over Na₂SO₄ and filtered. The filtrate wasconcentrated under reduced pressure to give a crude product, which waschromatographed eluting with 30% EtOAc/hexane to give 12.4 g of thetitle compound as a colorless oil.

7D. (2S,3R)-3-Hydroxy-2-pyrrolidinecarboxylic acid methyl ester,trifluoroacetic acid salt

[0253]

[0254] To a solution of 7D (12 g, 48.8 mmol) in CH₂Cl₂ (300 mL) cooledat 0° C. was added TFA (60 mL). After stirring at 0° C. for 30 min,additional amount of TFA (30 mL) was added. The mixture was allowed tostir at 0° C. for an additional 1 h, then concentrated and the oilyresidue stripped with diethylether (2×) under reduced pressure and driedin vacuo to give a white foam.

7E. (2S,3R)-3-Hydroxy-2-pyrrolidinecarboxylic acid methyl ester

[0255]

[0256] To a solution of 7D (48.8 mmol) in MeOH (120 mL) was addedDiaion® WA21J resin (70 g). The resulting suspension was stirred at roomtemperature for 20 min, and then filtered. The filtrate was concentratedcarefully under reduced pressure to give the title compound (7.7 g) as acolorless oil.

7F.(2S,3R)-1-(4-Cyano-naphthalen-1-ylcarbamoyl)-3-hydroxy-pyrrolidine-2carboxylicacid methyl ester

[0257]

[0258] To a solution of 7D (510 mg, 1.97 mmol) in CH₂Cl₂ (10 mL) cooledat 0° C. was added N,N-diisopropylethylamine (0.35 mL, 2 mmol) and themixture was stirred at RT for 20 min, then 6B (345 mg, 1.785 mmol) wasadded. After stirring for 30 min, the reaction was partitioned betweenCH₂Cl₂ and saturated aqueous NH₄Cl. The CH₂Cl₂ phase was washed withbrine, dried over Na₂SO₄ and filtered. The filtrate was concentratedunder reduced pressure to give a crude product, which waschromatographed (silica gel) eluting with 0.5% MeOH in EtOAc/hexane(1:1) to give 436 mg of the title compound as a white foam. MS (ES): m/z340 [M+1]⁺.

7G.(7R,7aS)-4-(Tetrahydro-7-hydroxy-1,3-dioxo-1H-pyrrolo[1,2-c]imidazol-2(3H)-yl)-1-naphthalenecarbonitrile

[0259] The title compound can also be prepared directly from 7E in anone-pot reaction as described below.

[0260] To a suspension of the compound 6B (10.67 g, 55 mmol) in toluene(500 mL) was added compound, 7E (7.7 g, 53 mmol), along with 4 Åmolecular sieves (˜20 g), the resulting mixture was stirred at RT untilformation of 7F was completed. To the mixture was added additional 400mL of toluene, followed by DBU (6.8 mL, 45.5 mmol), the resulting browncolored suspension was vigorously stirred at RT until the reaction wascompleted (3 hours). The reaction mixture was loaded on a silica gelcolumn, then eluted with 30% EtOAc/hexane, 50% EtOAc/hexane, and 5% MeOHin EtOAc/hexane (1:1) to give 8.6 g of 7G as an off-white solid (opticalpurity=98%), which was crystallized from isopropanol to yield 7.35 g ofthe title compound with 99.8% optical purity. mp. 235-237° C.; [α]_(D)^(rt)=−31; HPLC: 99% at 12.45 min (retention time) (CHIRALPAK® OD column4.6×250 mm; 30% isopropanol in hexane over 20 minutes, 1 mL/min,monitoring at 220 nM); MS (ES): m/z 308 [M+1]⁺.

EXAMPLE 8(7S,7aR)-4-(Tetrahydro-7-hydroxy-1,3-dioxo-1H-pyrrolo[1,2-c]imidazol-2(3H)-yl)-1-naphthalenecarbonitrile

[0261]

[0262] The title compound (1.47 g) was isolated from 6C (5.8 g) by usingpreparative HPLC with a chiral column (CHIRALPAK® AD, 5×50 cm, 20μ) and25% of isopropanol/hexane as an eluate. mp. 237-238° C.; [α]_(D)^(rt)=+29.76; HPLC: 99% at 10.77 min (retention time) (CHIRALPAK® ODcolumn 4.6×250 mm; 30% isopropanol in hexane over 20 minutes, 1 mL/min,monitoring at 220 nM); MS (ES): m/z 308 [M+1]⁺.

EXAMPLE 9trans-Tetrahydro-7-hydroxy-2-(4-nitro-1-naphthalenyl)-1H-pyrrolo[1.2-c]imidazole-1,3(2H)-dione

[0263]

[0264] The title compound (racemate) was prepared from 1A and 6A by theprocedures analogous to those described in Example 6C. mp. 255-257° C.;HPLC: 99% at 2.13 to 2.27 min (retention time) (YMC S5 ODS column4.6×50mm; 10-90% aqueous methanol over 4 minutes containing 0.2%phosphoric acid, 4mL/min, monitoring at 220 nM), MS (ES): m/z 328[M+1]⁺.

EXAMPLE 10(7S,7aR)-Tetrahydro-7-hydroxy-2-(4-nitro-1-naphthalenyl)-1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione

[0265]

[0266] 34.8 mg of the tile compound was separated from 80 mg of compound9 by using preparative HPLC with a chiral column (CHIRALPAK® AD, 5×50cm, 20μ) and 25% of isopropanol/hexane as an eluate. mp. 253-255° C.;HPLC: 99% at 14.95 min (retention time) (CHIRALPAK® AD column 4.6×250mm; 25% isopropanol in hexane over 20 minutes, 1 mL/min, monitoring at220 nM); MS (ES): m/z 328 [M+1]⁺

EXAMPLE 11(7R,7aS)-Tetrahydro-7-hydroxy-2-(4-nitro-1-naphthalenyl)-1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione

[0267]

[0268] 21 mg of the tile compound was separated from 80 mg of compound 9by using preparative HPLC with a chiral column (CHIRALPAK® AD, 5×50 cm,20μ) and 25% of isopropanol/hexane as an eluate. mp. 234-236° C.; HPLC:99% at 17.79 min (retention time) (CHIRALPAK® AD column 4.6×250 mm; 25%isopropanol in hexane over 20 minutes, 1 mL/min, monitoring at 220 nM);MS (ES): m/z 328 [M+1]⁺.

EXAMPLE 12trans-2-(4-Bromo-1-naphthalenyl)tetrahydro-7-hydroxy-1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione

[0269]

12A. 4-Bromo-1-naphthaleneisocyanate

[0270]

[0271] Compound 12A was prepared from 1-amino-4-bromonaphthalene in asimilar fashion to that described in Example 1A.

12B. trans-2-(4-Bromo-1-naphthalenyl)tetrahydro-7-hydroxy-1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione

[0272]

[0273] Compound 12B (racemate) was prepared from 6A and 12A byprocedures analogous to those described in Example 6C. mp. 204-206° C.;HPLC: 99% at 2.57-2.65 min (retention time) (YMC S5 ODS column 4.6×50mm;10-90% aqueous methanol over 4 minutes containing 0.2% phosphoric acid,4 mL/min, monitoring at 220 nM), MS (ES): m/z 362 [M+1]⁺.

EXAMPLE 13trans-4-(Tetrahydro-7-hydroxy-1,3-dioxo-1H-pyrrolo[1,2-c]imidazol-2(3H)-yl)-1-naphthalenecarboxylicacid methyl ester

[0274]

13A. 4-Nitro-1-naphthyldiazonium tetrafluoroborate salt

[0275]

[0276] To boron trifluorodiethyletherate (0.51 mL, 4.0 mmol) cooled at−20° C. was slowly added 1-amine-4-nitronaphthalene (500 mg, 3.0 mmol)in dimethoxy-ethylene (10 mL) over 10 minutes, followed by addition oftert-butylnitrite (0.42 mL, 3.55 mmol) over 1 minute. After addition,the reaction mixture was stirred at −20° C. for 10 minutes, then at 5°C. for 20 minutes and pentane (40 mL) was added. The resultingsuspension was filtered and the collected solid was washed with ether togive the title compound 13A (730 mg).

13B. 4-Nitro-1-naphthalenecarboxylic acid methyl ester

[0277]

[0278] To a suspension of compound 13A (620 mg, 2.16 mmol) in MeOH (50mL) cooled at 0° C. under argon was added palladium(II) acetate (24 mg,0.11 mmol). The reaction vessel was then charged with carbon monoxidegas. The reaction mixture was stirred at 0° C., then at RT. Theresulting homogenous reaction was quenched with water, extracted withCH₂Cl₂. The organic layer was separated, washed with brine, dried overNa₂SO₄ and concentrated under reduced pressure to provide a crudeproduct, which was chromatographed (silica gel) eluting with 2%EtOAc/hexane to give compound 13B (150 mg).

13C. 4-Amino-1-naphthalenecarboxylic acid methyl ester

[0279]

[0280] Compound 13B (92 mg, 0.398 mmol) and 10% Pd/C (10 mg) in MeOH (5mL) was hydrogenated under a hydrogen balloon for 30 minutes. Themixture was filtered and the filtrate concentrated under reducedpressure to give the title compound (80 mg).

13D. 4-Isocyano-1-naphthalenecarboxylic acid methyl ester

[0281]

[0282] Compound 13D was prepared from 13C by procedures analogous tothose described in Example 1A.

13E:trans-4-(Tetrahydro-7-hydroxy-1,3-dioxo-1H-pyrrolo[1,2-c]imidazol-2(3H)-yl)-1-naphthalenecarboxylicacid methyl ester

[0283] The title compound (racemate) was prepared from 13D by proceduresanalogous to those described in Example 6C. HPLC: 99% at 2.09-2.27 min(retention time) (YMC S5 ODS column 4.6×50mm; 10-90% aqueous methanolover 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at220 nM), MS (ES): m/z 341 [M+1]⁺

EXAMPLE 14trans-4-(Tetrahydro-7-methoxy-1,3-dioxo-1H-pyrrolo[1,2-c]imidazol-2(3H)-yl)-1-naphthalenecarbonitrile

[0284]

[0285] To a solution of compound 6C (100 mg, 0.326 mmol) in acetonitrile(2 mL), at RT, was added iodomethane (86 μL, 1.38 mmol), followed bysilver(I) oxide (42 mg, 0.18 mmol). The mixture was refluxed at 85° C.for 16 hours. After cooling to RT, the reaction mixture was filtered andthe filtrate was concentrated under reduced pressure to give a crudeproduct, which was purified by using a preparative HPLC (gradient, 20 to90% of MeOH /H₂O containing 0.1% of CF₃CO₂H over 10 minutes) to yieldthe title compound (racemate) (72 mg, 69% yield) as an off-white solid.mp. 185-186° C. HPLC: 99% at 2.45 min (retention time) (YMC S5 ODScolumn 4.6×50mm; 10-90% aqueous methanol over 4 minutes containing 0.2%phosphoric acid, 4 mL/min, monitoring at 220 nM), MS (ES): m/z 322[M+1]⁺.

EXAMPLE 154-(7-Acetoxy-1,3-dioxo-tetrahydro-pyrrolo[1,2-c]imidazol-2-yl)-naphthalene-1-carbonitrile

[0286]

[0287] To a solution of 6C (123 mg, 0.4 mmol) in pyridine (3 mL) wasadded acetic anhydride (0.26 mL, 2.8 mmol), followed by4-dimethylaminopyridine (15 mg). The reaction mixture was stirred at RTfor 3 h, then quenched with water and extracted with CH₂Cl₂ (3×10 mL).The combined organic extracts were washed with brine, dried over Na₂SO₄and filtered. The filtrate was concentrated under reduced pressure togive a crude product, which was chromatographed (silica gel) elutingwith 2% MeOH in EtOAc/hexane (1:1) to give 70 mg of the title compound(racemate) as a white solid. m.p. 105° C.; HPLC: 99% at 4.8 min(retention time) (Conditions: Zorbax SB C18 (4.6×75 mm); Eluted with 0%to 100% B, 8 min gradient. (A=90% H₂O—10% MeOH—0.1% H₃PO₄ and B=10%H₂O—90% MeOH—0.1% H₃PO₄); Flow rate at 2.5 mL/min. UV detection at 220nm). MS (ES): m/z 350 [M+1]⁺.

EXAMPLE 164-(7-Fluoro-1,3-dioxo-tetrahydro-pyrrolo[1,2-c]midazol-2-yl)-naphthalene-1-carbonitrile

[0288]

[0289] To a solution of diethylaminosulfur trifluoride (0.073 mL, 0.45mmol) in CH₂Cl₂ (2 mL) cooled at −78° C. in a flame-dried flask underargon was added dropwise a solution of 6C (92 mg, 0.3 mmol) in CH₂Cl₂(12 mL). After addition, the reaction mixture was stirred at −78° C. for30 min, then at 0° C. overnight. The reaction mixture was partitionedbetween CH₂Cl₂ and saturated aqueous NaHCO₃. The separated aqueous layerwas extracted with CH₂Cl₂ (2×). The combined CH₂Cl₂ layers were washedwith brine, dried over Na₂SO₄ and filtered. The filtrate wasconcentrated under reduced pressure to give a crude product, which waschromatographed (silica gel) eluting with EtOAc/hexane (1:2) to give 52mg of the title compound (racemate) as a white solid. m.p. 228-230° C.;HPLC: 99% at 5.0 min (retention time) (Conditions: Zorbax SB C18 (4.6×75mm); Eluted with 0% to 100% B, 8 min gradient. (A=90% H₂O—10% MeOH—0.1%H₃PO₄ and B=10% H₂O—90% MeOH—0.1% H₃PO₄); Flow rate at 2.5 mL/min. UVdetection at 220 nm). MS (ES): m/z 641 [2M+Na]⁺.

EXAMPLE 17(4-(5,7-Dioxo-dihydro-imidazo[1,5-c]thiazol-6-yl)-naphthalene-1-carbonitrile

[0290]

17A. Thiazolidine-4-carboxylic acid methyl ester

[0291]

[0292] Hydrogen chloride gas was bubbled through a suspension ofL-thioproline (990 mg, 7.55 mmol) in MeOH (30 mL) cooled at 0° C. for 5min. The resulting clear solution was stirred at RT for 2 h, thenconcentrated carefully under reduced pressure. The obtained residue wasdried in vacuo overnight to give a white solid, which was dissolved inMeOH (30 mL) and treated with Diaion® WA21J resin (6 g). The resultingsuspension was stirred at RT for 30 min, then filtered. The filtrate wasconcentrated carefully under reduced pressure and the oily residuestripped with toluene (2×) to give a colorless oil.

17B.(4-(5,7-Dioxo-dihydro-imidazo[1,5-c]thiazol-6-yl)-naphthalene-1carbonitrile

[0293] To a suspension of 17A (1.1 g, 7.6 mmol) and 4 Å molecular sieves(5 g) in toluene (40 mL) under argon was added a solution of 6B (1.47 g,76 mmol) in toluene (20 mL). After addition, the reaction mixture wasstirred at RT overnight, then DBU (1.05 mL, 7.55 mmol) was added. Theresulting dark red solution was stirred at 80° C. for 3 h. After coolingto RT, the reaction mixture was partitioned between CH₂Cl₂ and H₂O. Theorganic phase was washed with brine, dried over Na₂SO₄ and filtered. Thefiltrate was concentrated under reduced pressure to give a yellowishresidue, which was chromatographed (silica gel) eluting withEtOAc/hexane (4:7) to give 226 mg of the title compound (racemate) as awhite solid. m.p. 223-225° C.; HPLC: 99% at 5.2 min (retention time)(Conditions: Zorbax SB C18 (4.6×75 mm); Eluted with 0% to 100% B, 8 mingradient. (A=90% H₂O—10% MeOH—0.1% H₃PO₄ and B=10% H₂O—90% MeOH—0.1%H₃PO₄); Flow rate at 2.5 mL/min. UV detection at 220 nm). MS (ES): m/z641 [2M+Na]⁺.

EXAMPLE 184-(2,5,7-Trioxo-dihydro-imidazo[1,5-c]thiazol-6-yl)-naphthalene-1-carbonitrile

[0294]

[0295] To a solution of 17B (62 mg, 0.2 mmol) in CH₂Cl₂ (1.5 mL) wasadded 3chloroperoxybenzoic acid (46 mg, 0.2 mmol). The reaction wasstirred at RT for 2 h, then quenched with 5% aqueous Na₂S₂O₃ andextracted with CH₂Cl₂ (2×) and EtOAc (2×). The combined organic extractswere washed with saturated aqueous NaHCO₃, brine, dried over Na₂SO₄ andfiltered. The filtrate was concentrated under reduced pressure to give acrude product, which was chromatographed (silica gel) eluting withEtOAc/hexane (2:3) to give 6 mg of the title compound (reacemate) as awhite solid. HPLC: 97% at 4.0 min (retention time) (Conditions: ZorbaxSB C18 (4.6×75 mm); Eluted with 0% to 100% B, 8 min gradient. (A=90%H₂O—10% MeOH—0.1% H₃PO₄ and B=10% H₂O—90% MeOH—0.1% H₃PO₄); Flow rate at2.5 mL/min. UV detection at 220 nm). MS (ES): m/z 326 [M+1]⁺.

EXAMPLE 194-(2,5,7-Trioxo-dihydro-imidazo[1,5-c]thiazol-6-yl)-naphthalene-1-carbonitrile(racemate)

[0296]

[0297] The crude product from Example 18 was chromatographed (silicagel) eluting with EtOAc/hexane (2:3) to give 20 mg of the title compound(racemate) as a white solid. HPLC: 96% at 3.9 min (retention time)(Conditions: Zorbax SB C18 (4.6×75 mm); Eluted with 0% to 100% B, 8 mingradient. (A=90% H₂O—10% MeOH—0.1% H₃PO₄ and B=10% H₂O—90% MeOH—0.1%H₃PO₄); Flow rate at 2.5 mL/min. UV detection at 220 nm). MS (ES): m/z326 [M+1]⁺.

EXAMPLE 204-(1,3-Dioxo-5,6-dihydro-1H-pyrrolo[1,2-c]imidazol-2-yl)-naphthalene-1-carbonitrile

[0298]

[0299] To a suspension of compound 8 (200 mg, 0.65 mmol) in CH₂Cl₂ (3mL) was added pyridine (0.06 mL), followed by triflic anhydride (0.12mL, 0.71 mmol) and 4dimethylaminopyridine (5 mg). The reaction wasstirred at RT for 15 min and the resulting reddish clear solution wastreated with DBU (0.1 mL, 0.71 mmol). The mixture was stirred at RT for30 min, then quenched with saturated aqueous NH₄Cl solution andextracted with CH₂Cl₂ (3×). The combined extracts were washed with H₂O,brine, dried over Na₂SO₄ and filtered. The filtrate was concentratedunder reduced pressure to give a crude product, which waschromatographed (silica gel) eluting with EtOAc/hexane (1:1) to give 150mg of the title compound as an off-white solid. m.p. 230-232° C. HPLC:99% at 2.43 min (retention time) (Conditions: YMC. S5 ODS column (4.6×50mm); Eluted with 0% to 100% B, 4 min gradient. (A=90% H₂O—10% MeOH—0.1%H₃PO₄ and B=10% H₂O—90% MeOH—0.1% H₃PO₄); Flow rate at 4 mL/min. UVdetection at 220 nm). MS (ES): m/z 290 [M+1]⁺.

EXAMPLE 21(7aS)-4-(1,3-Dioxo-5,7a-dihydro-1H-pyrrolo[1,2-c]midazol-2-yl)-naphthalene-1-carbonitrile

[0300]

21A. 3,4-Dehydro-L-proline methyl ester, trifluoroacetic acid salt

[0301]

[0302] To a solution of N-Boc-3,4-dehydro-L-proline methyl ester(Peninsula Labs) (1 g, 4.405 mmol) in CH₂Cl₂ (3 mL) cooled at 0° C. wasadded TFA (2 mL). After addition, the reaction mixture was stirred at RTfor 1 h, then concentrated under reduced pressure to give an oilyresidue, which was stripped with ether (2×) and dried in vacuo to give acolorless oil (954 mg).

21B.(7aS)-4-(1,3-Dioxo-5,7a-dihydro-1H-pyrrolo[1,2-c]imidazol-2-yl)naphthalene-1-carbonitrile

[0303] To a solution of 21A (0.17 g, 0.75 mmol) in CH₂Cl₂ (5 mL) wasadded N,N diisopropylethylamine (0.135 mL, 0.77 mmol), followed by asolution of 6B (115 mg, 0.595 mmol) in CH₂Cl₂ (5 mL) and 4 Å molecularsieves (2 g). After stirring at RT for 3 h, DBU (0.1 mL, 0.65 mmol) wasadded, followed by toluene (10 mL) and the reaction mixture was heatedat 75° C. for 6 h. After cooling to RT, the reaction mixture waspartitioned between saturated aqueous NH₄Cl and CH₂Cl₂. The organiclayer was washed with brine, dried over Na₂SO₄ and filtered. Thefiltrate was concentrated under reduced pressure to give a crudeproduct, which was chromatographed (silica gel) eluting with 30%EtOAc/hexane to give a racemic product. The title compound (37 mg) wasisolated by using preparative HPLC with a chiral column (CHIRALPAK® AD,5×50 cm, 20μ) and 40% of isopropanol/hexane as an eluate. HPLC: 99% at19.4 min (retention time) (CHIRALPAK® AD column 4.6×250 mm; 35%isopropanol in hexane over 30 minutes, 1 mL/min, monitoring at 220 nM);MS (ES): m/z 290 [M+1]⁺.

EXAMPLE 22(7aR)-4-(1,3-Dioxo-5,7a-dihydro-1H-pyrrolo[,2-c]imidazol-2-yl)-naphthalene-1-carbonitrile

[0304]

[0305] The title compound (46 mg) was isolated from the racemateprepareded in Example 21 by using preparative HPLC with a chiral column(CHIRALPAK® AD, 5×50 cm, 20μ) and 40% of isopropanol/hexane as aneluate. HPLC: 99% at 13.2 min (retention time) (CHIRALPAK® AD column4.6×250 mm; 35% isopropanol in hexane over 30 minutes, 1 mL/min,monitoring at 220 nM); MS (ES): m/z 290 [M+1]⁺.

EXAMPLE 23(7aR)-4-(6,7-Dihydroxy-1,3-dioxo-tetrahydro-pyrrolo[1,2-c]imidazol-2-yl)-naphthalene-1-carbonitrile

[0306]

[0307] To a solution of compound 22 (50 mg, 0.173 mmol) in acetone/H₂O(5mL/0.2 mL) cooled at 0° C. was added 4-methylmorpholine N-oxide (41mg, 0.346 mmol), followed by osmium tetraoxide (2.5 wt. % solution in2-methyl-2-propanol) (0.353 mL, 0.035 mmol). The reaction mixture wasstirred at RT overnight, then filtered. The filtrate was concentratedunder reduced pressure to give a crude product, which was partitionedbetween EtOAc and saturated aqueous NaHCO₃. The organic layer was washedwith brine, dried over Na₂SO₄ and filtered. The filtrate wasconcentrated in vacuo and the residue chromatographed (silica gel)eluting with 10% MeOH in EtOAc/hexane (3:2) to give 44 mg of the titlecompound (diastereomeric mixture) as a white solid. m.p. 246° C. HPLC:99% at 2.02 min (retention time) (Conditions: YMC S5 ODS column (4.6×50mm); Eluted with 0% to 100% B, 4 min gradient. (A=90% H₂O—10% MeOH—0.1%H₃PO₄ and B=10% H₂O—90% MeOH—0.1% H₃PO₄); Flow rate at 4 mL/min. UVdetection at 220 nm). MS (ES): m/z 324 [M+1]⁺.

EXAMPLE 24(7aS)-4-(6,7-Dihydroxy-1,3-dioxo-tetrahydro-pyrrolo[1,2-c]imidazol-2-yl)-naphthalene-1-carbonitrile

[0308]

[0309] The title compound (diastereomeric mixture) (52 mg) was preparedfrom compound 21 (50 mg) by the procedures analogous to those describedin Example 23. m.p. 225° C. HPLC: 99% at 2.03 min (retention time)(Conditions: YMC S5 ODS column (4.6×50 mm); Eluted with 0% to 100% B, 4min gradient. (A=90% H₂O—10% MeOH—0.1% H₃PO₄ and B=10% H₂O—90% MeOH—0.1%H₃PO₄); Flow rate at 4 mL/min. UV detection at 220 nm). MS (ES): m/z 324[M+1]⁺.

EXAMPLE 25(7S,7aR)-4-(7-Hydroxy-7a-methyl-1,3-dioxo-tetrahydro-pyrrolo[1,2-c]imidazol-2-yl)-naphthalene-1carbonitrile

[0310]

[0311] To a stirring suspension of compound 8 (115 mg, 0.374 mmol) inTHF (3 mL) at RT under argon was added drops of DMPU. The resultingclear solution was cooled at −78° C., then LDA (2 M solution inTHF/heptane/ethylbenzene) (0.37 mL, 0.74 mmol) was added dropwise. Theresulting dark brown solution was stirred at −78° C. for 25 min, theniodomethane (0.069 mL, 0.74 mmol) was added. After addition, thereaction mixture was allowed to warm up to −5 to 0° C., and stirred at−5 to 0° C. for 4 h. The reddish colored reaction mixture was quenchedwith 5% aqueous KHSO₄ and extracted with EtOAc (2×20 mL). The combinedorganic extracts were washed with brine, dried over Na₂SO₄ and filtered.The filtrate was concentrated in vacuo and the residue chromatographed(silica gel) eluting with 3% MeOH in EtOAc/hexane (1:1) to give 57 mg ofthe product, which was further purified using preparative HPLC with achiral column (CHIRALPAK® AD, 5×50 cm, 20 ) and 20% isopropanol/hexaneas an eluate to give 30 mg of the title compound. m.p. 234-235° C.Chiral HPLC: 99% at 13.9 min (retention time) (CHIRALPAK® AD column4.6×250 mm; 20% isopropanol in hexane over 30 minutes, Imumin,monitoring at 220 nM); MS (ES): m/z 322 [M+1]⁺.

EXAMPLE 26(7R,7aS)-4-(7-Hydroxy-7a-methyl-1,3-dioxo-tetrahydro-pyrrolo[1,2-c]imidazol-2-yl)-naphthalene-1-carbonitrile

[0312]

[0313] Compound 26 was prepared from 7G by the procedures analogous tothose described in Example 25. m.p. 232-234° C. Chiral HPLC: 99% at 24min (retention time) (CHIRALPAK® AD column 4.6×250 mm; 20% isopropanolin hexane over 30 minutes, 1 mL/min, monitoring at 220 nM); MS (ES): m/z322 [M+1]⁺.

EXAMPLE 27(7R,7aS)-4-(7-Hydroxy-7-methyl-1,3-dioxo-tetrahydro-pyrrolo[1,2-c-]imidazol-2-yl)-naphthalene-1-carbonitrile

[0314]

27A. [tert-Butoxycarbonyl-(3-oxo-butyl)-amino]-acetic acid ethyl ester

[0315]

[0316] The title compound was prepared by the procedures described inStocking, E. M.; et al, J. Am. Chem. Soc. 122, 1675-1683 (2000).

27B. 3-Hydroxy-3-methyl-pyrrolidine-1,2-dicarboxylic acid 1-tert-butylester 2-ethyl ester

[0317]

[0318] To a solution of 27A (5 g, 18.29 mmol) in toluene (100 mL) cooledat 0° C. was added solid potassium tert-butoxide (2.05 g, 18.29 mmol) insmall portions to maintain the solution temperature <5° C. The reactionwas stirred at 0° C. for 45 min, then quenched by addition of ice-cooled10% aqueous KHSO₄ solution and extracted with CH₂Cl₂ (3×). The combinedorganic extracts were washed with brine, dried over Na₂SO₄ and filtered.The filtrate was concentrated under reduced pressure to give a brownoily residue, which was chromatographed (silica gel) eluting withEtOAc/hexane (1:3) to give 530 mg of the title compound (racemate).

27C.(7R,7aS)-4-(7-Hydroxy-7-methyl-1,3-dioxo-tetrahydro-pyrrolo[1,2c]imidazol-2-yl)-naphthalene-1-carbonitrile

[0319] To a solution of 27B (500 mg, 1.83 mmol) in CH₂Cl₂ (1.5 mL) wasadded TFA (1.5 mL). The reaction mixture was stirred at RT for 1 h, thenconcentrated under reduced pressure to give an oily residue, which wasstripped with toluene (3×) and dried in vacuo overnight. The oilyresidue was dissolved in CH₂Cl₂ (10 mL) and to the solution was added N,N-diisopropylethylamine (0.35 mL, 2.01 mmol), followed by a solution of6B (355 mg, 1.83 mmol) in CH₂Cl₂ (5 mL) and 4 Å molecular sieves (1 g).The reaction mixture was stirred at RT for 1 h, the resulting turbidsolution (formation of the urea intermediate) was treated with DBU (0.41mL, 2.745 mmol), and stirred at RT for an additional 1 h. The clearreaction solution was partitioned between CH₂Cl₂ and 5% aqueous KHSO₄.The aqueous phase was extracted with CH₂Cl₂ (2×) and the combined CH₂Cl₂extracts were washed with brine, dried over Na₂SO₄ and filtered. Thefiltrate was concentrated under reduced pressure to give a crudeproduct, which was chromatographed (silica gel) eluting with 4% MeOH inEtOAc/hexane (1:1) to give 330 mg of the desired product as a racemicmixture. The title compound (8 mg) was isolated by using preparativeHPLC with a chiral column (CHIRALPAK® AD, 5×50 cm, 20μ) and 10% ofisopropanol/hexane as an eluate. Chiral HPLC: 99% at 20.6 min (retentiontime) (CHIRALPAK® AD column 4.6×250 mm; 20% isopropanol in hexane over30 minutes, 1 mL/min, monitoring at 220 nM); MS (ES): m/z 322 [M+1]⁺.

EXAMPLE 28(7S,7aR)-4-(7-Hydroxy-7-methyl-1,3-dioxo-tetrahydro-pyrrolo[1,2-c]imidazol-2-yl)-naphthalene-1-carbonitrile

[0320]

[0321] The title compound (7 mg) was isolated from the racemateprepareded in Example 27C by using preparative HPLC with a chiral column(CHIRALPAK® AD, 5×50 cm, 20μ) and 10% of isopropanol/hexane as aneluate. Chiral HPLC: 99% at 23.5 min (retention time) (CHIRALPAK® ADcolumn 4.6×250 mm; 20% isopropanol in hexane over 30 minutes, 1 mL/min,monitoring at 220 nM); MS (ES): m/z 322 [M+1]⁺.

EXAMPLE 29(7R,7aS)-4-(7-Hydroxy-1-oxo-3-thioxo-tetrahydro-pyrrolo[1,2-c]imidazol-2-yl)-naphthalene-1-carbonitrile

[0322]

29A. 4-Cyano-N-thionylnaphthylamine

[0323]

[0324] To a suspension of 4-cyano-naphthylamine (200 mg, 1.19 mmol) andNaHCO₃ (1 g) in CHCl₃ (15 mL) at RT was added thiophosgene (2.36 mL,30.8 mmol) dropwise. After addition, the reaction mixture was stirred atRT for 4 h, then filtered and the filtrate concentrated in vacuo todryness to give the title compound as a solid.

29B.(7R,7aS)-4-(7-Hydroxy-1-oxo-3-thioxo-tetrahydro-pyrrolo[1,2-c]imidazol-2-yl)-naphthalene-1-carbonitrile

[0325] To a solution of 7D (325 mg, 1.25 mmol) in CH₂Cl2 (5 mL) wasadded N,N-diisopropylethylamine (0.32 mL, 1.875 mmol). The reactionmixture was stirred at RT for 30 min, then a solution of 29A in toluene(10 mL) was added. After stirring at RT for 2 h, DBU (0.185 mL, 1.20mmol) was added and the reaction mixture was stirred for an additional 2h. The reaction mixture was concentrated in vacuo and the residuechromatographed (silica gel) eluting with 30% EtOAc in hexane to give267 mg as a white solid, which was further purified using preparativeHPLC with a chiral column (CHIRALPAK® AD, 5×50 cm, 20μ) and 30%isopropanol/hexane as an eluate to give 50 mg of the title compound as awhite solid. m.p. 215-216° C. Chiral HPLC: 98% at 19.1 min (retentiontime) (CHIRALPAK® AD column 4.6×250 mm; 30% isopropanol in hexane over30 minutes, 1 mL/min, monitoring at 220 nM); MS (ES): m/z 324 [M+1]⁺.

EXAMPLE 30(3R,7S,7aS)-4-(3-tert-Butyl-7-hydroxy-1-oxo-tetrahydro-pyrrolo[1,2-c]midazol-2-yl)-naphthalene-1-carbonitrile

[0326]

30A. (2S,3S)-3-Hydroxy-pyrrolidine-1,2-dicarboxylic acid 1-tert-butylester

[0327]

[0328] A solution of trans-3-hydroxy-L-proline (9.0 g, 69.2 mmol) indioxane (94 ml) and water (46 ml) was cooled to 0° C., treated withsodium hydroxide (1.77 g, 44.3 mmol) followed by di-tert-butyldicarbonate (16.74 g, 7.67 mmol). The mixture was stirred at 0° C. for 5min, at RT for 6 h then quenched with 2.0 N hydrochloric acid (40 ml, 80mmol) and extracted with EtOAc (3×350 ml). The combined organic extractswere washed with brine (45 ml) and dried to provide the title compoundas a white solid (15.6 g, 97.3%). MS (ES): m/z 254 [M+Na]⁺.

30B.(2S,3S)-3-(tert-Butyl-dimethyl-silanyloxy)-pyrrolidine-1,2-dicarboxylicacid 1-tert-butyl ester

[0329]

[0330] A cooled (0° C.) solution of 30A (15.0 g, 64.8 mmol) andimidazole (22.0 g, 324 mmol) in dry DMF (110 ml) was treated withtert-butyldimethylsilyl chloride (24.3 g, 162 mmol), warmed to RT andstirred for 24 h. The reaction mixture was treated with methanol (150ml), stirred at RT for another 24 h then partitioned between 10% aqueouscitric acid (225 ml) and EtOAc (3×1.0 L). The extracts were washed withbrine (200 ml), dried (Na₂SO₄) and filtered. The filtrate wasconcentrated to dryness to give a thick colorless syrup (46.0 g).Purification of a portion (20 g) by using automated flash chromatography(135 g silica gel column, EtOAc/hexane gradient) gave the title compoundas a thick clear syrup (9.7 g, 99.7%).

30C.(2S,3S)-3-(tert-Butyl-dimethyl-silanyloxy)-2-(4-cyano-naphthalen-1-ylcarbamoyl)-pyrrolidine-1-carboxylicacid tert-butyl ester

[0331]

[0332] A solution of isobutylchloroformate (0.75 ml, 5.8 mmol) in dryEtOAc (8.0 ml) was cooled to −20° C., treated dropwise with a mixture of30B (2.0 g, 5.8 mmol) and N-methylmorpholine (0.64 ml, 5.8 mmol) in dryEtOAc (6.0 ml) over a period of 45 minutes. The mixture was stirred at−20° C. for 30 minutes, then treated dropwise over a period of 1.5 hwith a solution of 4-aminonaphthalene carbonitrile (976 mg, 5.8 mmol) indry EtOAc (35 ml). The reaction mixture was stirred at −20° C. for anadditional 3.0 h, then at RT for 4 days after which it was partitionedbetween water (50 ml) and EtOAc (3×250 ml). Purification by flashchromatography provided the title compound as a dark pink foam (2.19 g,76.3%). MS (ES): m/z 496 [M+1]⁺.

30D. (2S,3S) 3-Hydroxy-pyrrolidine-2-carboxylic acid(4-cyano-naphthalen-1-yl)amide

[0333]

[0334] A solution of 30C (700 mg, 1.4 mmol) in dry CH₂Cl₂ (2.8 ml) andTFA (2.8 ml) was stirred at RT for 4 days. The reaction mixture wasdiluted with CH₂Cl₂ (35 ml), concentrated to a syrup and stripped withtoluene (35 ml) and ether (2×20 ml). The residual syrup was dissolved inwater (7.0 ml), basified with 1.0 N NaHCO₃ (5.0 ml) to pH 8.0, extractedwith CH₂Cl₂ (2×100 ml) and the combined organic extracts were dried(Na₂SO₄) and concentrated. Purification by automated flashchromatography (35 g silica gel column, EtOAc/hexane gradient) gave thetitle compound as an off-white solid (249 mg, 83.4%), m.p. 174-176° C.MS (ES): m/z 282 [M+1]⁺.

30E.(3R,7S,7aS)-4-(3-tert-Butyl-7-hydroxy-1-oxo-tetrahydro-pyrrolo[1,2c]imidazol-2-yl)-naphthalene-1-carbonitrile

[0335] A suspension of 30D (40 mg, 0.14 mmol) and pivalaldehyde (30 ul,0.27 mmol) in a mixture of dry benzene (0.5 ml) and dry pentane (5.0 ml)was refluxed for 20 hr with a Dean-Starke receiver, after whichadditional pivalaldehyde (30 ul, 0.27 mmol), dry benzene (0.5 ml) andcatalytic toluenesulfonic acid was added (Ref: Org. Lett. 2 (18)2781-2783, (2000)). The reaction mixture was refluxed for another 25 hr,evaporated to dryness and dried in vacuo. Purification using automatedflash chromatography (10 g silica gel column, EtOAc/hexane gradient)gave the title compound (single diastereoisomer) as a white foam (40 mg,81.8%). MS (ES): m/z 350 [M+1]⁺

EXAMPLE 31(3R,7S,7aS)-4-(7-Hydroxy-1-oxo-3-phenyl-tetrahydro-pyrrolo[1,2-c]imidazol-2-yl)-naphthalene-1-carbonitrile

[0336]

[0337] The title compound was obtained from 30D and benzaldehyde in thesame manner as described in Example 30E as a cream-colored foam (17.1mg, 33.1%) by separation on a chiral column (CHIRALPAK® AD, 5×50 cm,20μ) and 30% isopropanol/hexane as an eluate. MS (ES): m/z 370 [M+1]⁺.

EXAMPLE 32(3S,7S,7aS)-4-(7-Hydroxy-1-oxo-3-phenyl-tetrahydro-pyrrolo[1,2-c]imidazol-2-yl)-naphthalene-1-carbonitrile

[0338]

[0339] The title compound was obtained from 30D and benzaldehyde in thesame manner as described in Example 30E, as an off-white solid (21.8 mg,42.2%), by separation on a chiral column (CHIRALPAK® AD, 5×50 cm, 20μ)and 30% isopropanol/hexane as an eluate. MS (ES): m/z 370 [M+1]⁺.

EXAMPLE 33(3R,6R,7aS)-4-(3-tert-Butyl-6-hydroxy-1-oxo-tetrahydro-pyrrolo[1,2-c]imidazol-2-yl)-naphthalene-1-carbonitrile

[0340]

33A.(2S,4R)-4-Benzyloxy-2-(4-cyano-naphthalen-1-ylcarbamoyl)-pyrrolidine-1-carboxylicacid tert-butyl ester

[0341]

[0342] A solution of 1-[tert-butoxy)carbonyl]-4-R-benzyloxy-L-proline(300 mg, 0.934 mmol) in dry THF (8.0 ml) was cooled to −15° C., treatedwith N-methylmorpholine (0.12 ml, 1.12 mmol) followed byisobutylchloroformate (0.132 ml, 1.03 mmol). The whitish suspension wasstirred at −15° C. for 30 min, treated with 4-aminonaphthalenecarbonitrile (157.3 mg, 0.934 mmol) and stirred at −15° C. for 30 minthen at room temperature for 4 days. (Ref: J. Med. Chem., 33, 635-645,(1998)). The reaction mixture was partitioned between water (7.0 ml) andEtOAc (3×35 ml) and the combined organic extracts washed with brine (7.0ml) and dried (Na₂SO₄). Purification using automated flashchromatography (35 g silica gel column, EtOAc/hexane gradient) providedthe title compound as a clear syrup (201.7 mg, 45.8 %). MS (ES): m/z 472[M+1]⁺.

33B. (2S,4R)-4-Benzyloxy-pyrrolidine-2-carboxylic acid(4-cyano-naphthalen-1-yl)-amide

[0343]

[0344] A solution of 33A (201 mg, 0.43 mmol) in dry CH₂Cl₂ (0.6 ml) andTFA (0.6 ml) was stirred at RT for 40 min. The reaction mixture wasdiluted with CH₂Cl₂ (5 ml), concentrated to a syrup and chased withtoluene (2×10 ml) and ether (2×50 ml) to give a syrup (202.9 mg).

[0345] The residual syrup (185 mg, 0.38 mmol) was dissolved in water(2.0 ml), basified with 1.0 N NaHCO₃ (0.45 ml), extracted with CH₂Cl₂(2×20 ml) and the combined organic extracts were washed with brine (2.0ml), dried (Na₂SO₄) and concentrated in vacuo to give the title compoundas a light beige syrup (151 mg, 100 %). MS (ES): m/z 372 [M+1]⁺.

33C.(3R,6R,7aS)-4-(6-Benzyloxy-3-tert-butyl-1-oxo-tetrahydro-pyrrolo[1,2-c]imidazol-2-yl)-naphthalene-1-carbonitrile

[0346]

[0347] A suspension of 33B (151 mg, 0.38 mmol) and pivalaldehyde (100μl, 0.90 mmol) in a mixture of dry benzene (0.5 ml) and dry pentane (5.0ml) was refluxed for 25 hr with a Dean-Starke receiver (Ref: Org. Lett.,2 (18) 2781-2783, (2000)). The reaction mixture was evaporated todryness and dried in vacuo. Purification using automated flashchromatography (35 g silica gel column, EtOAc/hexane gradient) gave thetitle compound as a light pink-colored solid (144.1 mg, 86.3%). m.p.155-157° C. MS (ES): m/z 440 [M+1]⁺

33D.(3R,6R,7aS)-4-(3-tert-Butyl-6-hydroxy-1-oxo-tetrahydro-pyrrolo[1,2-c]imidazol-2-yl)-naphthalene-1-carbonitrile

[0348] Compound 33C (94.4 mg, 0.2 mmol) was dissolved in dry CH₂Cl₂ (5.0ml), cooled to −75° C., treated with 1.0 M BCl₃/CH₂Cl₂ (1.2 ml, 1.2mmol) and stirred at −75° C. for 1.5 hr. The reaction mixture wasquenched with ice-water (3.4 ml), warmed to room temperature, stirredfor 5 min then extracted with CH₂Cl₂ (50 ml). The organic phase waswashed with water (3.4 ml) and brine (3.4 ml) and dried (Na₂SO₄).Purification using automated flash chromatography (35 g silica gelcolumn, EtOAc/hexane gradient) gave the title compound as a white solid(34.7 mg, 49.6%), m.p. 155-157° C. MS (ES): m/z 350[M+1]⁺.

EXAMPLE 344-(8-Hydroxy-1,3-dioxo-hexahydro-imidazo[1,5-a]pyridin-2-yl)-naphthalene-1-carbonitrile

[0349]

34A. cis-3-Hydroxy-piperidine-2-carboxylic acid

[0350]

[0351] A suspension of 3-hydroxypicolinic acid (1 g, 7.19 mmol) and 5wt. % rhodium on carbon (200 mg) in a mixed solvents of conc. NH₄OH (35mL) and water (5 mL) in a pressure bottle was stirred under hydrogen at80 psi overnight. The reaction mixture was filtered and the filtrateconcentrated in vacuo to dryness to give 118 mg of the title compound(racemate). MS (ES): m/z 146 [M+1]⁺.

34B. cis-3-Hydroxy-piperidine-2-carboxylic acid methyl ester;hydrochloride salt

[0352]

[0353] Hydrogen chloride gas was bubbled into a suspension of 34A (1.18g, 7.19 mmol) in MeOH (100 mL) for 5 min. The resulting clear solutionwas stirred at RT for 1 h, then concentrated carefully under reducedpressure to give a crude product, which was stripped with toluene (2×)and dried in vacuo to give the title compound (1.4 g).

34C.4-(8-Hydroxy-1,3-dioxo-hexahydro-imidazo[1,5-a]pyridin-2-yl)naphthalene-1-carbonitrile

[0354] To a suspension of 34B (353 mg, 1.81 mmol) and 4 A molecularsieves (2 g) in CH₂Cl₂ (5 mL) was added N,N-diisopropylethylamine (0.32mL, 1.875 mmol). The reaction mixture was stirred at RT for 30 min, thena solution of 6B (275 mg, 1.41 mmol) in toluene (10 mL) was added. Afterstirring for 2 h, DBU (0.198 mL, 1.41 mmol) was added and the reactionmixture was stirred at RT overnight. The reaction mixture waspartitioned between EtOAc and saturated aqueous NH₄Cl solution. Theseparated organic layer was concentrated in vacuo and the residuechromatographed (silica gel) eluting with EtOAc/hexane (1:1), then 3%MeOH in EtOAc/hexane (1:1) to give 230 mg of the title compound(racemate) as a white solid. mp. 243° C. MS (ES): m/z 322 [M+1]⁺.

EXAMPLE 35(8R,8aS)-4-(8-Hydroxy-1,3-dioxo-hexahydro-imidazo[1,5-a]pyridin-2-yl)-naphthalene-1-carbonitrile

[0355]

[0356] The title compound (31 mg) was isolated from racemic 34C (49 mg)using preparative HPLC with a chiral column (CHIRALPAK® AD, 5×50 cm,20μ) and 30% isopropanol/hexane as an eluate as a white solid. m.p. 243°C. Chiral HPLC: 99% at 16.5 min (retention time) (CHIRALPAK® AD column4.6×250 mm; 30% isopropanol in hexane over 30 minutes, 1 mL/min,monitoring at 220 nM); MS (ES): m/z 322 [M+1]⁺.

EXAMPLE 36(8S,8aR)-4-(8-Hydroxy-1,3-dioxo-hexahydro-imidazo[1,5-a]pyridin-2-yl)-naphthalene-1-carbonitrile

[0357]

[0358] The title compound (12 mg) was isolated from racemic 34C (49 mg)using preparative HPLC with a chiral column (CHIRALPAK® AD, 5×50 cm,20μ) and 30% isopropanol/hexane as an eluate as a white solid. m.p. 244°C. Chiral HPLC: 99% at 21.1 min (retention time) (CHIRALPAK® AD column4.6×250 mm; 30% isopropanol in hexane over 30 minutes, lmUmin,monitoring at 220 nM); MS (ES): m/z 322 [M+1]⁺.

EXAMPLE 372-(4-Cyano-naphthalen-1-yl)-1,3-dioxo-hexahydro-imidazo[1,5-a]pyrazine-7carboxylicacid tert-butyl ester

[0359]

[0360] The title compound (930 mg) was prepared fromN-4-tert-butoxycarbonyl-2-piperazinecarboxylic acid tert-butyl ester(859 mg) by procedures analogous to those described in Experiment 6C asa white solid. m.p. 222-224° C. Chiral HPLC: 2 peaks at 13.5 min (52%)and 17.6 min (48%) (CHIRALPAK® AS column 4.6×250 mm; 25% isopropanol inhexane over 30 minutes, 1 mL/min, monitoring at 220 nM); MS (ES): m/z407 [M+1]⁺.

EXAMPLE 384-(1,3-Dioxo-hexahydro-imidazo[1,5-a]pyrazin-2-yl)-naphthalene-1-carbonitrile,hydrochloride salt

[0361]

[0362] To a solution of 37 (90 mg, 0.22 mmol) in CH₂Cl₂ (3 mL) was added4 N HCl/dioxane (0.44 mL, 1.76 mmol). After addition, the reactionmixture was stirred at RT for 2 h and a white precipitate formed. Thereaction was filtered and the precipitate was triturated with hexane(2×10 mL). The gummy residue was dissolved in water and lyophilized togive 46 mg of the title compound (racemate) as a white powder. m.p. 200°C. HPLC: 97% at 2.9 min (retention time) (Conditions: Zorbax SB C18(4.6×75 mm); Eluted with 0% to 100% B, 8 min gradient. (A=90% H₂O—10%MeOH—0.1% H₃PO₄ and B=10% H₂O—90% MeOH—0.1% H₃PO₄); Flow rate at 2.5mL/min. UV detection at 220 nm). MS (ES): m/z 307 [M+1]⁺.

EXAMPLE 394-(7-Methanesulfonyl-1,3-dioxo-hexahydro-imidazo[1,5-a]pyrazin-2-yl)naphthalene-1-carbonitrile

[0363]

[0364] To a solution of 38 (70 mg, 0.164 mmol) in CH₂Cl₂ (2 mL) at 0° C.was added methanesulfonyl chloride (0.014 mL, 0.18 mmol), followed byEt₃N (0.114 mL, 0.82 mmol). After addition, the reaction mixture wasstirred at 0-5° C. for 30 min, then loaded on a silica gel cartridgeeluting EtOAc/hexane gradient (automated flash chromatography) to give46 mg of the title compound (racemate) as a white solid. m.p. 207-216°C. HPLC: 100% at 4.4 min (retention time) (Conditions: Zorbax SB C18(4.6×75 mm); Eluted with 0% to 100% B, 8 min gradient. (A=90% H₂O—10%MeOH—0.1% H₃PO₄ and B=10% H₂O—90% MeOH—0.1% H₃PO₄); Flow rate at 2.5mL/min. UV detection at 220 nm). MS (ES): m/z 385 [M+1]⁺.

EXAMPLE 402-(4-Cyano-naphthalen-1-yl)-1,3-dioxo-hexahydro-imidazo[1,5-a]pyrazine-7-carboxylicacid methyl ester

[0365]

[0366] To a solution of 38 (70 mg, 0.164 mmol) in CH₂Cl₂ (2 mL) at 0° C.was added methyl chloroformate (0.015 mL, 0.2 mmol), followed by Et₃N(0.114 mL, 0.82 mmol). After addition, the reaction mixture was stirredat 0-5° C. for 30 min, then loaded on a silica gel cartridge elutingEtOAc/hexane gradient (auto flash chromatography) to give 42 mg of thetitle compound (racemate) as a white powder. HPLC: 100% at 4.8 min(retention time) (Conditions: Zorbax SB C18 (4.6×75 mm); Eluted with 0%to 100% B, 8 min gradient. (A=90% H₂O—10% MeOH—0.1% H₃PO₄ and B=10%H₂O—90% MeOH—0.1% H₃PO₄); Flow rate at 2.5 mL/min. UV detection at 220nm). MS (ES): m/z 365 [M+1]⁺.

EXAMPLE 41(7R,7aR)-4-(7-Hydroxy-3-oxo-tetrahydropyrrolo[1,2-c]imidazol-2-yl)naphthalene-1-carbonitrile

[0367]

41A.(7R,7aS)-4-(1,7-Dihydroxy-3-oxo-tetrahydropyrrolo[1,2-c]imidazol-2-yl)-naphthalene-1-carbonitrile

[0368]

[0369] To a suspension of 7G (307 mg, 1.0 mmol) in anhydrous THF (3 mL)at RT was added DMPU (0.5 mL). The resulting solution was cooled at −78°C., then a solution of 1.0 M LiEt₃BH/THF (2.5 mL, 2.5 mmol) was addeddropwise. After stirring at −78° C. for 1 h, then at 40° C. for 16 h,the reaction mixture was quenched with saturated aqueous NaHCO₃ at 40°C. The mixture was stirred at 0° C. for 30 min, then treated with H₂O₂and extracted with CH₂Cl₂ (3×). The combined CH₂Cl₂ extracts were washedwith brine, dried over Na₂SO₄ and filtered. The filtrate wasconcentrated under reduced pressure to give a crude product, which waschromatographed (silica gel) eluting with 3-5% MeOH in CH₂Cl₂ to givethe title compound as a white solid (190 mg). MS (ES): m/z 310 [M+1]⁺.

41B.(7R,7aR)-4-(7-Hydroxy-3-oxo-tetrahydropyrrolo[1,2-c]imidazol-2-yl)-naphthalene-1-carbonitrile

[0370] To a suspension of 41A (190 mg, 0.615 mmol) in anhydrous CH₂Cl₂(3 mL) at RT was added DMPU (0.5 mL). The resulting solution was cooledat −78° C., then Et₃SiH (0.8 mL, 5.0 mmol) was added, followed by BF₃Et₂O (0.75 mL, 6.0 mmol). The reaction mixture was stirred at −78° C.for 3 h, then at −40° C. overnight. The reaction mixture was quenchedwith saturated aqueous NaHCO₃ at −40° C. The mixture was stirred at RTfor 30 min, then extracted with CH₂Cl₂ (3×). The combined CH₂Cl₂extracts were washed with brine, dried over Na₂SO₄ and filtered. Thefiltrate was concentrated under reduced pressure to give a crude oilyproduct, which was purified using preparative HPLC to give a white solid(75 mg). The white solid was crystallized from hot isopropyl alcohol toyield the title compound as a white solid (48 mg as a pure singleisomer). mp 226-228° C. Chiral HPLC: 99% at 10.20 min (retention time)(CHIRALPAK® AD column 4.6×250 mm; 40% isopropanol in hexane over 20minutes, 1 mL/min, monitoring at 220 nM); MS (ES): m/z 294 [M+1]⁺.

EXAMPLE 42(7S,7aS)-4-(7-Hydroxy-3-oxo-tetrahydropyrrolo[1,2-c]imidazol-2-yl)-naphthalene-1-carbonitrile

[0371]

42A.(7S,7aR)-4-(1,7-Dihydroxy-3-oxo-tetrahydropyrrolo[1,2-c]imidazol-2-yl)-naphthalene-1-carbonitrile

[0372]

[0373] The title compound (176 mg) was prepared from compound 8 (370 mg)by procedures analogous to those described in Example 41A. MS (ES): m/z310 [M+1]⁺.

42B.(7S,7aS)-4-(7-Hydroxy-3-oxo-tetrahydropyrrolo[1,2-c]imidazol-2-yl)-naphthalene-1-carbonitrile

[0374] The title compound (40 mg) was prepared from compound 42A (176mg) by procedures analogous to those described in Example 41B. mp226-228° C. Chiral HPLC: 99% at 7.36 min (retention time) (CHIRALPAK® ADcolumn 4.6×250 mm; 40% isopropanol in hexane over 20 minutes, 1 mL/min,monitoring at 220 nM); MS (ES): m/z 294 [M+1]⁺.

What is claimed is:
 1. A compound of the formula Ia

wherein R₁ is selected from the group consisting of hydrogen (H), cyano(—CN), nitro (—NO₂), halo, heterocyclo, OR₄, CO₂R₅, CONHR₅, COR₅,S(O)_(m)R₅, SO₂NR₅R₅′, NHCOR₅ and NHSO₂R₅; R₂ is selected from the groupconsisting of hydrogen (H), alkyl or substituted alkyl, alkenyl orsubstituted alkenyl, arylalkyl or substituted arylalkyl, CO₂R₅, CONR₅R₅′and CH₂OR₅; R₃ is selected from the group consisting of hydrogen (H),alkyl or substituted alkyl, alkenyl or substituted alkenyl, alkynyl orsubstituted alkynyl, cycloalkyl or substituted cycloalkyl, arylalkyl orsubstituted arylalkyl, heterocycloalkyl or substituted heterocycloalkyl,aryl or substituted aryl, heteroaryl or substituted heteroaryl, halo,cyano(—CN), NHCOR₅, NHCO₂R₅, NHCONR₅R₅′, NHSO₂R₅ and OR₄; R₄ in eachfunctional group is independently selected from the group consisting ofhydrogen (H), alkyl or substituted alkyl, CHF₂, CF₃ and COR₅; R₅ and R₅′in each functional group are each independently selected from the groupconsisting of hydrogen(H), alkyl or substituted alkyl, alkenyl orsubstituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl orsubstituted cycloalkyl, heterocycloalkyl or substitutedheterocycloalkyl, arylalkyl or substituted arylalkyl, aryl orsubstituted aryl, heteroaryl or substituted heteroaryl and —CN; W isselected from the group consisting of (CR₆R₆′)_(m), CHOH(CR₆R₆′)_(m),CO(CR₆R₆′)_(m) and C═NOR₄(CR₆R₆′)_(m); R₆ and R₆′ are each independentlyselected from the group consisting of hydrogen (H), alkyl or substitutedalkyl, alkenyl or substituted alkenyl, alkynyl or substituted alkynyl,cycloalkyl or substituted cycloalkyl, arylalkyl or substitutedarylalkyl, heterocycloalkyl or substituted heterocycloalkyl, aryl orsubstituted aryl, heteroaryl or substituted heteroaryl, halo, cyano(—CN), NHCOR₅, NHCO₂R₅, NHCONR₅R₅′, NHSO₂R₅ and OR₄; X is selected fromthe group consisting of methylene (—CH₂—), oxygen(O), S(O)_(m), NCOR₅,NCO₂R₅, NCONHR₅R₅′ NSO₂NR₅R₅′; Y is selected from the group consistingof oxygen(O), sulfur(S) and hydrogen (H₂); Z is selected from the groupconsisting of oxygen(O), sulfur(S), NH and NR₅; A and B are eachindependently selected from the group consisting of hydrogen (H), halo,cyano(—CN), nitro(—NO₂), alkyl or substituted alkyl and OR₄; m is aninteger from 0 to 2; and n is an integer of 1 or 2; including allprodrug esters, pharmaceutically acceptable salts and stereoisomersthereof.
 2. A pharmaceutical composition comprising a compound asdefined in claim
 1. 3. The pharmaceutical composition of claim 2 furthercomprising at least one additional therapeutic agent selected from thegroup consisting of parathyroid hormone, bisphosphonates, estrogen,testosterone, progesterone, selective estrogen receptor modulators,growth hormone secretagogues, growth hormone, progesterone receptormodulators, anti-diabetic agents, anti-hypertensive agents,anti-inflammatory agents, anti-osteoporosis agents, anti-obesity agents,cardiac glycosides, cholesterol lowering agents, anti-depressants,anti-anxiety agents, anabolic agents, and thyroid mimetics.
 4. A methodfor treating or delaying the progression or onset of muscular atrophy,lipodistrophy, long-term critical illness, sarcopenia, frailty orage-related functional decline, reduced muscle strength and function,reduced bone density or growth, the catabolic side effects ofglucocorticoids, chronic fatigue syndrome, bone fracture repair, acutefatigue syndrome and muscle loss following elective surgery, cachexia,chronic catabolic state, eating disorders, side effects of chemotherapy,wasting, depression, nervousness, irritability, stress, growthretardation, reduced cognitive function, male contraception,hypogonadism, Syndrome X, diabetic complications or obesity, whichcomprises administering to a mammalian species in need of treatment atherapeutically effective amount of a compound as defined in claim
 1. 5.A method according to claim 4 further comprising administering,concurrently or sequentially, a therapeutically effective amount of atleast one additional therapeutic agent selected from the groupconsisting of parathyroid hormone, bisphosphonates, estrogen,testosterone, progesterone, selective estrogen receptor modulators,growth hormone secretagogues, growth hormone, progesterone receptormodulators, anti-diabetic agents, anti-hypertensive agents,anti-inflammatory agents, anti-osteoporosis agents, anti-obesity agents,cardiac glycosides, cholesterol lowering agents, anti-depressants,anti-anxiety agents, anabolic agents, and thyroid mimetics.